Living Colors User Manualcoli.usal.es/web/abydl/biblioteca/bibelectro.alu/... · 1999. 2. 10. · B. Expresion of GFP Fusion Proteins 18 C. GFP Expression in Mammalian Cells 20 D

Living Colors TM

User Manual

(PT2040-1)

FOR RESEARCH USE ONLY

(PR74631)

CLONTECH Laboratories, Inc.

page Protocol # PT2040-1 Technical Service TEL:415-424-8222 or 800-662-CLON2 Version # PR74631 FAX: 415-424-1064 or 800-424-1350

Table of Contents

I. Introduction 4

II. Properties of GFP and GFP Variants 6A. The GFP Chromophore 6B. Red-shifted GFP Variants 7C. Blue Emission GFP Variants 8D. UV-optimized GFP Variant 8E. Acquisition and Stability of GFP Fluorescence 10F. Sensitivity 13

III. Living Colors GFP and GFP Variant Vectors 14

IV. Expression of GFP and GFP Variants 16A. Suitable Host Organisms and Cells 16B. Expresion of GFP Fusion Proteins 18C. GFP Expression in Mammalian Cells 20D. GFP Expression in Plants 21E. GFP Expression in Yeast 22

V. Detection of GFP and GFP Variants 23A. Filter Sets 23B. Flow Cytometry 24C. Fluorescence Microscopy 24D. Quantitative Fluorometric Assay 26

VI. Purified Recombinant GFP and GFP Variants 29A. General Information 29B. rGFP and rGFP Variants as Standards in Western Blots 29C. rGFP and rGFP Variants as a Control for

Fluorescence Microscopy 31VII. GFP Antibodies 32

A. Applications for GFP Antibodies 32B. Immunoprecipitation with GFP Monoclonal Antibody 32

VIII. Troubleshooting Guide 34

IX. References 38

X. Summary Reference List 46

XI. Related Products 48

Appendix. Fluorescent Proteins Newsgroup 50


TEL:415-424-8222 or 800-662-CLON Technical Service Protocol # PT2040-1 pageFAX:415-424-1064 or 800-424-1350 Version # PR74631 3

I. Introduction & Protocol Overview

List of Figures

Figure 1. The amino acid substitutions of the red-shifted GFPvariants and EBFP 6

Figure 2. Excitation and emission spectra of wt GFP and red-shiftedGFP variants 7

Figure 3. Excitation and emission spectra of wt GFP and EBFP 9Figure 4. Excitation and emission spectra of wt GFP and GFPuv 9Figure 5. Comparison of fluorescence intensity of GFP in sonication

buffer versus GFP in cell lysates 26Figure 6. Relative fluorescence intensity of serial dilutions of

GFP-transformed cells 28

List of Tables

Table I. Living Colors Vectors 15Table II. Proteins Expressed as Fusions to GFP 18–19Table III. Use of GFP in Plants 21



In the bioluminescent jellyfish Aequorea victoria, light is produced when energyis transferred from the Ca2+-activated photoprotein aequorin to green fluorescentprotein (GFP; Shimomura et al., 1962; Morin & Hastings, 1971; Ward et al.,1980). The cloning of the wild-type GFP gene (wt GFP; Prasher et al., 1992;Inouye & Tsuji, 1994a) and its subsequent expression in heterologous systems(Chalfie et al., 1994; Inouye & Tsuji, 1994a; Wang & Hazelrigg, 1994) hasestablished GFP as a novel genetic reporter system. When expressed in eithereukaryotic or prokaryotic cells and illuminated by blue or UV light, GFP yields abright green fluorescence. Light-stimulated GFP fluorescence is species-independent and does not require any cofactors, substrates, or additional geneproducts from A. victoria. Additionally, detection of GFP and its variants can beperformed with living tissues instead of fixed samples.

CLONTECH offers many GFP products, including expression vectors, sequenc-ing primers, purified Recombinant GFP Proteins, and GFP-specific Monoclonaland Polyclonal Antibodies. Also available from CLONTECH are several vectorsencoding GFP variants that were developed to improve the use of GFP as areporter for gene expression and protein localization. These variants are ideal forfluorescence microscopy and flow cytometry. (See Section III for further informa-tion on specific vectors and Section XI for a complete list of related products.)

• Red-shifted GFP variants: EGFP (GFPmut1; Cormack et al., 1996) andGFP-S65T (Heim et al., 1995) have red-shifted excitation spectra andfluoresce 4–35-fold more brightly than wt GFP when excited with blue light.

• EBFP: The blue fluorescent variant, EBFP, contain four amino acid substitu-tions that shift the emission from green to blue, enhance the brightness of thefluorescence, minimize rapid photobleaching, and improve solubility of theprotein (Heim & Tsien, 1996; Cormack et al., 1996).

• GFPuv: When expressed in E. coli, GFPuv is reported to be 18 times brighterthan wt GFP when excited by standard UV light (Crameri et al., 1996) andshould be used in experiments using UV light for excitation. This variantcontains additional amino acid mutations which also increase the translationalefficiency of the protein in E. coli.

In addition to the chromophore mutations mentioned above, the GFP variantgenes hGFP-S65T, EGFP, and EBFP contain more than 190 silent mutationswhich create an open reading frame comprised almost entirely of preferredhuman codons (Haas, et al., 1996). Furthermore, in the construction of the LivingColors vectors containing these variant genes, upstream sequences flanking thecoding regions were converted to a Kozak consensus translation initiation site(Kozak, 1987), and potentially inhibitory flanking sequences in the original cDNAclones (λgfp10 & pGFP10.1; Chalfie et al., 1994) were removed. All of thesechanges increased the translational efficiency of the mRNA—and, conse-quently, the expression of the GFP variants—in mammalian and plant systems.

I. Introduction



The development of EGFP, GFPuv, and more recently EBFP, opens up a widerange of exciting new applications that were not possible with GFP alone. Thecombined use of EBFP and wt GFP, EGFP, GFPuv, or one of the other greenGFP variants, should permit the following types of double-labeling experiments:1) microscopy of multiple cell populations in a mixed culture; 2) monitoring geneexpression from two different promoters in the same cell, tissue, or organism;3) the intracellular localization of two different proteins (Rizzuto et al., 1996);4) the monitoring of different cell lineages in a single tissue or organism; 5) thereal-time analysis of interactions between two distinct protein fusions (Heim &Tsien, 1996; Mitra et al., 1996); and 6) FACS® analysis of mixed cell populations(e.g., a mixture of cells expressing EGFP and EBFP or GFPuv, and nonfluorescentcells). GFPuv may be used in double-labeling experiments with EGFP byselective excitation of these two variants.

The GFP Application Notes include information about GFP and GFP variants;protocols for the expression and detection of GFP and its variants; suggestedapplications for the Recombinant GFP Proteins and GFP-specific antibodies;and a comprehensive list of GFP references.

I. Introduction continued



II. Properties of GFP and GFP Variants

A. The GFP ChromophoreThe GFP chromophore consists of a cyclic tripeptide derived from Ser-Tyr-Gly in the primary protein sequence (Cody et al., 1993) and is onlyfluorescent when embedded within the complete GFP protein. (There havebeen no reports of a truncated GFP [other than a few amino acids from theC-terminus] that is still fluorescent.) The crystal structures of GFP and GFP-S65T have revealed a tightly packed β-can enclosing an α-helix containingthe chromophore (Ormö et al., 1996; Yang, F. et al., 1996). This structureprovides the proper environment for the chromophore to fluoresce byexcluding solvent and oxygen. Nascent GFP is not fluorescent, sincechromophore formation occurs posttranslationally. The chromophore isformed by a cyclization reaction and an oxidation step that requiresmolecular oxygen (Heim et al., 1994; Davis et al., 1995). These steps areeither autocatalytic or use factors that are ubiquitous, since fluorescentGFP forms in a broad range of organisms. Chromophore formation may bethe rate-limiting step in generating the fluorescent protein, especially ifoxygen is limiting (Heim et al., 1994; Davis et al., 1995). The wt GFPabsorbs UV and blue light with a maximum peak of absorbance at 395 nmand a minor peak at 470 nm and emits green light maximally at 509 nm, witha shoulder at 540 nm (Ward et al., 1980).

Figure 1. The amino acid substitutions (in grey) of the red-shifted GFP variants and EBFP areshown below the wt GFP sequence.

Phe 64 Ser Tyr Gly Val Gln 69. . . . Tyr 145

Leu 64 Thr Tyr Gly Val Gln 69

Leu 64 Thr His Gly Val Gln 69. . . . Phe145 Phe 64 Thr Tyr Gly Val Gln 69

wt GFP:

EGFP:

EBFP:

GFP-S65T:



II. Properties of GFP and GFP Variants continued

B. Red-Shifted GFP VariantsCLONTECH’s red-shifted GFP variants EGFP and phGFP-S65T containdifferent mutations in the chromophore (Figure 1), which shift the maximalexcitation peak to approximately 490 nm (Figure 2). The major excitationpeak of the red-shifted variants encompasses the excitation wavelength ofcommonly used filter sets, so the resulting signal from these variants ismuch brighter than from wt GFP. Similarly, the argon ion laser used in mostFACS machines and confocal scanning laser microscopes emits at 488 nm,so excitation of the red-shifted GFP variants is much more efficient thanexcitation of wt GFP. In practical terms, this means the detection limits inboth microscopy and FACS are considerably lower with the red-shiftedvariants. Although the peak positions in the emission spectra of all the red-shifted GFP variants are virtually identical, double-labeling can be per-formed by selective excitation of wt GFP and red-shifted GFP (Kain et al.,1995; Yang, T.T. et al., 1996b). It should also be possible to use EGFP andGFPuv in double-labeling experiments.

phGFP-S65T contains the Ser-65 to Thr mutation described by Heim et al.(1995; referred to as S65T in that publication). In addition, the GFP-S65Tgene variant present in phGFP-S65T contains more than 190 silent basechanges that correspond to human codon-usage preferences. These silent

Figure 2. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (grey lines) andred-shifted GFP variants (black lines). The emission data for wt GFP were obtained with excitation at475 nm. (The emission peak for wt GFP is several-fold higher when illuminated at 395 nm). The emissiondata for the red-shifted GFP were obtained by exciting GFP-S65T at 489 nm (Heim et al., 1995). The otherred-shifted GFP variants have similar spectra.

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changes increase the translational efficiency and, therefore, the expres-sion of GFP-S65T in mammalian systems (Haas et al., 1996). In side-by-side comparisons, GFP-S65T fluorescence is 4–6 times more intense thanwt GFP and has a single, red-shifted excitation peak at 490 nm. GFP-S65Talso acquires fluorescence about four times faster than wt GFP (Heim et al.,1995).

EGFP contains the double-amino-acid substitutions Phe-64 to Leu andSer-65 to Thr (previously published as GFPmut1; Cormack et al., 1996).Based on spectral analysis of equal amounts of soluble protein, GFPmut1fluoresces 35-fold more intensely than wt GFP when excited at 488 nm(Cormack et al., 1996; Yang, T. T. et al., 1996a), due to an increase in itsextinction coefficient (Em). The Em for EGFP has been measured at61,000 cm-1 M-1 at 488 nm excitation (D. W. Piston, pers. comm.), comparedwith 7,000 cm-1 M-1 for wt GFP, and 39,200 cm-1 M-1 for GFP-S65T (Heim etal., 1995) under similar conditions. (Data on the rate of formation of anactive fluorophore are not yet available for EGFP.) To ensure maximalmammalian expression, the EGFP gene also contains the human-codonoptimized silent base changes present in hGFP-S65T (Haas et al., 1996).

C. Blue Emission GFP VariantsThe EBFP variant, developed at CLONTECH, contains four amino acidsubstitutions. The Tyr-66 to His substitution gives EBFP fluorescenceexcitation and emission maxima (380 nm and 440 nm, respectively) similarto other blue emission variants (Figure 3; Heim & Tsien, 1996; Mitra et al,1996; Heim et al., 1994). The other three substitutions (Phe-64 to Leu; Ser-65 to Thr; and Tyr-145 to Phe; Figure 1) enhance the brightness andsolubility of the protein, primarily due to improved protein folding propertiesand efficiency of chromophore formation (Heim & Tsien, 1996; Cormack etal., 1996). In addition, the coding sequence of the EBFP gene contains thesame set of 190 silent base changes present in EGFP and, thus, is humancodon-optimized (Haas et al., 1996). The Em of EBFP is 31,000 cm–1 M–1

for 380-nm excitation, leading to a fluorescent signal that is 2–3-foldbrighter than other blue variants of GFP and roughly equivalent to wt GFP.

D. UV-Optimized GFP VariantGFPuv is optimized for maximal fluorescence when excited by UV light(360–400 nm) and for higher bacterial expression. The GFPuv variant isideal for experiments in which GFP expression will be detected using UVlight for chromophore excitation (e.g., for visualizing bacteria or yeastcolonies). GFPuv was developed at Maxigene by Crameri et al. (1996)using an in vitro DNA shuffling technique to introduce point mutations.

GFPuv contains three amino acid substitutions (Phe-99 to Ser, Met-153 toThr, and Val-163 to Ala [based on the amino acid numbering of wt GFP]),



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Figure 3. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (black lines) andEBFP (grey lines). The emission data were obtained with excitation at 390 nm.


Figure 4. Excitation (dashed lines) and emission (solid lines) spectra of wt GFP (black lines) andGFPuv (grey lines). The emission data were obtained with excitation at 385 nm. (Data derived fromCrameri et al., 1996.)

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none of which alter the chromophore sequence. These mutations makeE. coli expressing GFPuv fluoresce 18 times brighter than wt GFP (Crameriet al., 1996). While these mutations dramatically increase the fluorescenceof GFPuv through their effects on protein folding and chromophore forma-tion, the emission and excitation maxima remain at the same wavelengthsas those of wt GFP (Crameri et al., 1996; Figure 4). However, GFPuv hasa greater propensity to dimerize than wt GFP (see Section II.E.8).

GFPuv expressed in E. coli is a soluble, fluorescent protein even underconditions in which the majority of wt GFP is expressed in a nonfluorescentform in inclusion bodies. This GFP variant also appears to have lowertoxicity than wt GFP; hence, the E. coli containing GFPuv grow two to threetimes faster than those expressing wt GFP (Crameri et al., 1996). Further-more, the GFPuv gene is a synthetic GFP gene in which five rarely used Argcodons from the wt gene were replaced by codons preferred in E. coli.Consequently, the GFPuv gene is expressed very efficiently in E. coli.

E. Acquisition and Stability of GFP Fluorescence1. Protein solubility

In bacterial expression systems, much of the expressed wt GFP is foundin an insoluble, nonfluorescent form in inclusion bodies. In contrast, almostall EGFP and GFPuv is expressed as a soluble, fluorescent protein.

2. PhotobleachingGFP fluorescence is very stable in a fluorometer (Ward, pers. comm.).Even under the higher intensity illumination of a fluorescence micro-scope, GFP is more resistant to photobleaching than is fluorescein(Wang & Hazelrigg, 1994; Niswender et al., 1995). The fluorescence ofwt GFP, EGFP, GFP-S65T, and RSGFP is quite stable when illumi-nated with 450–490 nm light (the major excitation peak for the red-shifted variants, but the minor peak for wt GFP). Some photobleachingoccurs when wt GFP is illuminated near its major excitation peak with340–390 nm or 395–440 nm light (Chalfie et al., 1994; Niswender et al.,1995). The rate of photobleaching of wt GFP and its green variants alsovaries with the organism being studied; for example, GFP fluorescenceis quite stable in Drosophila (Wang & Hazelrigg, 1994) and zebrafish.In C. elegans, 10 mM NaN3 accelerates photobleaching (Chalfie et al.,1994). Studies of the photobleaching characteristics of GFPuv havenot been performed to date.

Rapid photobleaching has been a problem with previously describedblue variants of GFP; however, our results with EBFP-transfectedCHO-K1 cells indicate that the rate of photobleaching in this variant isone-half to one-third that of P4-3, a popular predecessor to EBFP whichcontains only two amino acid substitutions (Tyr-66 to His and Tyr-145to Phe; Heim & Tsien, 1996).



3. Stability to oxidation/reductionGFP needs to be in an oxidized state to fluoresce because chro-mophore formation is dependent upon an oxidation of Tyr-66 (Heim etal., 1994). Strong reducing agents, such as 5 mM Na2S2O4 or 2 mMFeSO4, convert GFP into a nonfluorescent form, but fluorescence isfully recovered after exposure to atmospheric oxygen (Inouye & Tsuji,1994b). Weaker reducing agents, such as 2% β-mercaptoethanol,10 mM dithiothreitol (DTT), 10 mM reduced glutathione, or 10 mML-cysteine, do not affect the fluorescence of GFP (Inouye & Tsuji,1994b). GFP fluorescence is not affected by moderate oxidizing agents(Ward, pers. comm.).

4. Stability to pHwt GFP retains fluorescence in the range pH 5.5-12; however, fluores-cence intensity decreases between pH 5.5 and pH 4, and drops sharplyabove pH 12 (Bokman & Ward, 1981). The EGFP and GFP-S65Tvariants exhibit a reduced range of pH stability. For these variants,fluorescence is stable between pH 7.0 and pH 11.5, drops sharplyabove pH 11.5, and decreases between pH.7.0 and pH 4.5, retainingabout 50% of fluorescence at pH 6.0 (Ward, pers. com.).

5. Stability to chemical reagentsGFP fluorescence is retained in mild denaturants, such as 1% SDS or8 M urea, and after fixation with glutaraldehyde or formaldehyde, butfully denatured GFP is not fluorescent. GFP is very sensitive to somenail polishes used to seal coverslips (Chalfie et al., 1994; Wang &Hazelrigg, 1994); therefore, use molten agarose or rubber cement toseal coverslips on microscope slides. Fluorescence is also quenchedby the nematode anesthetic phenoxypropanol (Chalfie et al., 1994).GFP fluorescence is irreversibly destroyed by 1% H2O2 and sulfhydrylreagents such as 1 mM DTNB (5,5'-dithio-bis-[2-nitrobenzoic acid])(Inouye & Tsuji, 1994b). Many organic solvents can be used atmoderate concentrations without abolishing fluorescence; however,the absorption maximum may shift (Robart & Ward, 1990).

6. Protein stabilityin vitro :GFP is exceptionally resistant to heat (Tm=70°C), alkaline pH, deter-gents, chaotropic salts, organic solvents, and most common pro-teases, except pronase (Bokman & Ward, 1981; Ward, 1981; Roth,1985; Robart & Ward, 1990). Some GFP fluorescence can be observedwhen nanogram amounts of protein are resolved on native or 1% SDSpolyacrylamide gels (Inouye & Tsuji, 1994a).





Fluorescence is lost if GFP is denatured by high temperature, extremesof pH, or guanidinium chloride, but can be partially recovered if theprotein is allowed to renature (Bokman & Ward, 1981; Ward & Bokman,1982). A thiol compound may be necessary to renature the protein intothe fluorescent form (Surpin & Ward, 1989).in vivo:GFP appears to be stable when expressed in various organisms,however, no measurement of its half-life has been reported.

7. Temperature sensitivity of GFP chromophore formation.Although fluorescent GFP is highly thermostable (Section II.E.6; Ward,1981), it appears that the formation of the GFP chromophore istemperature sensitive. In yeast, GFP fluorescence was strongest whenthe cells were grown at 15°C, decreasing to about 25% of this value asthe incubation temperature was raised to 37°C (Lim et al., 1995).However, GFP and GFP-fusions synthesized in S. cerevisiae, at 23°Cretain fluorescence despite a later shift to 35°C (Lim et al., 1995). It hasalso been noted that E. coli expressing GFP show stronger fluores-cence when grown at 24°C or 30°C compared to 37°C (Heim et al.,1994; Ward, pers. comm.). Mammalian cells expressing GFP havealso been seen to exhibit stronger fluorescence when grown at30–33°C compared to 37°C (Pines, 1995; Ogawa et al., 1995).EGFP and GFPuv contain mutations that increase the efficiency ofprotein folding and chromophore formation (Cormack et al ,1996,Crameri et al., 1996). The same mutations also suppress thethermosensitivity of chromophore formation; thus, EGFP and GFPuvshow little difference in fluorescence when expressed at either 25°C or37°C. One of the GFPuv mutations is also present in other GFP variantsselected for strong fluorescence at 37°C (Siemering et al, 1996).

8. Dimerization of GFPGFP is fluorescent either as a monomer or as a dimer. The ratio ofmonomeric and dimeric forms depends on the protein concentrationand environment. wt GFP dimerizes via hydrophobic interactions atprotein concentrations above 5–10 mg/ml and in high-salt conditions.Dimerization results in a 4-fold reduction in the absorption at 470 nmand a concomitant increase in absorption at 395 nm (Ward, pers. com.).GFPuv has a greater propensity to dimerize than wtGFP; the 470-nmexcitation peak is suppressed at protein concentrations about 5-foldlower than for wt GFP (Ward, pers. comm.). In most experimentalsystems using wt GFP as a reporter, the monomeric form will predomi-nate; however, when GFPuv is used as the reporter, dimerization maybe evident even at moderate expression levels.




F. Sensitivity1. General considerations

GFP, like fluorescein, has a quantum yield of about 80% (Ward, 1981),although the extinction coefficient for GFP is much lower. Neverthe-less, in fluorescence microscopy, GFP fusion proteins have been foundto give greater sensitivity and resolution than staining with fluorescentlylabeled antibodies (Wang & Hazelrigg, 1994). GFP fusions have theadvantages of being more resistant to photobleaching and of avoidingbackground caused by nonspecific binding of the primary and second-ary antibodies to targets other than the antigen (Wang & Hazelrigg,1994). Although binding of multiple antibody molecules to a singletarget offers a potential amplification not available for GFP, this is offsetbecause neither labeling of the antibody nor binding of the antibody tothe target is 100% efficient.The mutations present in the green variants GFPuv, EGFP, and GFP-S65T significantly increase the sensitivity of GFP as a reporter.Subcellular localization of GFP can further increase the sensitivity ofGFP by concentrating the signal to a small area within the cell.However, for some applications, the sensitivity of GFP may be limitedby autofluorescence or limited penetration of light. Studies with wt GFPexpressed in HeLa cells (Niswender et al., 1995) have shown that thecytoplasmic concentration must be greater than ~1.0 µM to obtain asignal that is twice the autofluorescence. This threshold for detectionis lower with the red-shifted variants: ~200 nM for GFP-S65T and~100 nM for EGFP (Piston, pers. comm.).The absolute sensitivity attainable with the blue variant EBFP isapproximately equivalent to that of wt GFP. However, relative to otherpublished blue variants (e.g., P4 and P4-3; Heim & Tsien, 1996), EBFPis brighter due to its higher extinction coefficient. At this time, we do nothave detailed information regarding the threshold amount of intracel-lular EBFP protein required to obtain a detectable signal.

2. As a quantitative reporterThe signal from GFP and its variants does not have any enzymaticamplification; hence, the sensitivity of GFP will probably be lower thanthat for enzymatic reporters, such as β-galactosidase, SEAP, andfirefly luciferase. However, GFP signals can be quantified by flowcytometry, confocal scanning laser microscopy, and fluorometricassays. Purified GFP and GFP variants can be quantified in a fluoro-meter-based assay in the low-nanogram range.



CLONTECH offers a variety of vectors (described below and in Table I) forexpression of GFP and its variants in many kinds of hosts. (See Section XI for acomplete listing of vectors and related products.) The vector information packetthat accompanies any purchased vector provides a vector map, multiple cloningsite (MCS) sequence, and additional information about the vector. Much of thisinformation is also available on the CLONTECH Web Site (http://www.clontech.com).Living Colors GFP and GFP variant vectors:

• pGFPuv is primarily intended as a source of the GFPuv gene. The gene canbe readily excised using restriction sites in the flanking MCS regions.Alternatively, the GFPuv coding sequence can be amplified by PCR.

• pBAD-GFPuv expresses the UV-optimized GFP (GFPuv) in bacterialsystems from the regulated arabinose promoter.

• pEGFP is primarily intended as a source of the EGFP gene. The gene canbe readily excised using restriction sites in the flanking MCS regions.Alternatively, the EGFP coding sequence can be amplified by PCR.

• pEGFP-1 is a transcrption reporter vector used for monitoring the activityof promoters or promoter/enhancer combinations cloned into the MCSupstream of the EGFP gene.

• pEGFP-N1, -N2, -N3 express a human codon-optimized, red-shifted GFPvariant and can be used for fusing heterologous proteins to the N-terminusof EGFP.

• pEGFP-C1, -C2, -C3 express a human codon-optimized, red-shifted GFPvariant and can be used for fusing heterologous proteins to the C-terminusof EGFP.

• pEBFP is primarily intended as a source of the EBFP gene. The gene canbe readily excised using restriction sites in the flanking MCS regions.Alternatively, the EBFP coding sequence can be amplified by PCR.

• pEBFP-N1 expresses a human codon-optimized, blue fluorescent GFPvariant and can be used for fusing heterologous proteins to the N-terminusof EBFP.

• pEBFP-C1 expresses a human codon-optimized, blue fluorescent GFPvariant and can be used for fusing heterologous proteins to the C-terminusof EBFP.

• pIRES-EGFP is used for the simultaneous expression, from one RNAtranscript, of EGFP and another recombinant protein of interest in trans-fected mammalian cells.

• phGFP-S65T expresses a human codon-optimized, red-shifted GFP vari-ant and can be used for fusing heterologous proteins to GFP-S65T.

• pGFP is primarily intended as a source of the wt GFP gene. The GFP genecan be readily excised using restriction sites in the flanking MCS regions.Alternatively, the GFP coding sequences can be amplified by PCR.

III. Living Colors GFP and GFP Variant Vectors



III. Living Colors GFP and GFP Variant Vectors continued

Sequencing PrimersThe GFP-N and -C Sequencing Primers can be used to sequence acrossthe junction of protein fusions to the N- or C-terminus of GFP, or to confirmthe identity or orientation of promoters inserted upstream of the GFP gene.Similarly, the EGFP-N and -C Sequencing Primers can be used with EGFP,EBFP, and hGFP-S65T.

TABLE I. LIVING COLORS VECTORS

GFP Fluorescence Excitation a Codon GenBankVector Variant Intensity Maxima (nm) Optimized Host Cells Accession #

pEGFP EGFP 35x 488 humanb prokaryoticd U76561

pEGFP-1 EGFP 35x 488 humanb mammaliand U55761

pEGFP-N1,2,3 EGFP 35x 488 humanb mammaliand U55762U57608U57609

pEGFP-C1,2,3 EGFP 35x 488 humanb mammaliand U55763U57606U57607

pEBFP EBFP n.a. 380 humanb mammaliand

pEBFP-N1, C1 EBFP n.a. 380 humanb mammaliand

phGFP-S65T GFP-S65T 4–6x 489 humanb mammaliand U43284

pGFPuv GFPuv 18x 395 E. coli c prokaryotice U62636

pBAD-GFPuv GFPuv 18x 395 E. coli c prokaryotice U62637

pGFP wild-type 1x 395 (470) none prokaryotic U17997

pIRES-EGFP EGFP 35x 488 humanb mammaliand

a The emission maximum for EBFP is 440 nm; for wt GFP and all other green variants, theemission maximum is 509 nm, with a shoulder at 540 nm.

b The GFP insert in these vectors contains >190 silent base mutations that correspond tohuman codon-usage preferences.

c Five rarely used arginine codons from the wt GFP gene have been replaced by codonspreferred in E. coli.

d Although optimized for mammalian expression, the GFP insert in these vectors can be clonedinto an appropriate vector for high-level expression of GFP in plants.

e Although optimized for bacterial expression, the GFP insert in these vectors should besuperior to wt GFP in any expression system using UV light for chromophore excitation.



A. Suitable Host Organisms and CellsAs mentioned in the Introduction (Section I), GFP fluorescence is species-independent. Indeed, fluorescence has been reported from many differenttypes of GFP-expressing hosts, including the following:

1. MicrobesAgrobacterium tumefaciens (Matthysse et al., 1996)Anabaena (Buikema, 1995)Candida albicans (Cormack et al., 1997)Dictyostelium (Hodgkinson, 1995; Maniak et al., 1995; Moores et al.,

1996; Fey et al., 1995)Escherichia coli (Chalfie et al., 1994; Inouye & Tsuji, 1994a; Burlage

et al., 1995; Kitts et al., 1995; Yu & van den Engh, 1995; Crameriet al., 1996)

Mycobacterium (Kremer et al., 1995)Polysphondylium pallidum (Fey et al., 1995)Pseudomonas putida (Matthysse et al., 1996)Rhizobium meliloti (Gage et al., 1996)Saccharomyces cerevisiae (Flach et al., 1994; Kahana et al., 1995;

Lim et al., 1995; Riles et al., 1995; Schlenstedt et al., 1995;Stearns, 1995; Halme et al., 1996)

Salmonella (Valdivia & Falco, 1996 )Schizosaccharomyces pombe (Nabeshima et al., 1995; Atkins &

Izant, 1995)Ustilago maydis (Spellig et al., 1996)Yersinia (Jacobi et al., 1995)

2. InvertebratesAedes aegypti (Higgs et al., 1996)Caenorhabditis elegans (Chalfie et al., 1994; Sengupta et al., 1994;

Treinin & Chalfie, 1995)Diamondback moth (Chao et al., 1996)Drosophila melanogaster (Wang & Hazelrigg, 1994; Barthmaier &

Fyrberg, 1995; Brand, 1995; Yeh et al., 1995)Lytechinus pictus (Seid et al., 1996)

3. Vertebrates293 cells (transformed primary embryonal kidney, human) (Marshall et

al., 1995)A-375 cells (malignant melanoma, human) (Levy et al., 1996)BHK cells (kidney, hamster) (Olson et al., 1995; Carey et al., 1996)BOSC23 cells (Cheng et al., 1996)

IV. Expression of GFP and GFP Variants



IV. Expression of GFP and GFP Variants continued

Chicken embryonic retina cells (Ogawa et al., 1995)CHO-K1 cells (ovary, Chinese hamster) (Kain et al., 1995; Kitts et al.,

1995; Olson et al., 1995; Yang, T.T. et al., 1996b; Crameri et al.,1996)

COS-1 cells (kidney, SV40 transformed, African green monkey)(Ogawa et al., 1995)

COS-7 cells (kidney, SV40 transformed, African green monkey)(Olson et al., 1995; Pines, 1995; Cheng et al., 1996; Haas et al.,1996)

EPC cells (epithelial, carp) (Ogawa et al., 1995)Ferret neurons (Lo et al., 1994)GH3 cells (pituitary tumor, rat) (Plautz et al., 1996)HeLa cells (epitheloid carcinoma, cervix, human) (Kaether & Gerdes,1995; Pines, 1995; Niswender et al., 1995)Hepatocytes (liver, rat) (Venerando et al., 1996)JEG cells (placenta, human) (Yu & Dong, 1995)NIH/3T3 cells (embryo, contact-inhibited, NIH Swiss mouse) (Olson etal., 1995; Pines, 1995)PA317 (embryo fibroblast, mouse) (Levy et al., 1996)PC-12 cells (adrenal pheochromocytoma, rat) (pers. comm)Pt K1 cells (kidney, kangaroo rat) (Olson et al., 1995)Mice (Ikawa et al., 1995a; Ikawa et al., 1995b)Xenopus (Tannahill et al., 1995; Wu et al., 1995)Zebrafish (Amsterdam et al., 1995)

4. PlantsArabidopsis thaliana (thale cress) (stable) (Haselhoff & Amos, 1995;Chiu et al., 1996; Reichel et al., 1996)Citrus sinensis (L.) Osbeck cv. Hamlin (an embryogenic sweet-orangecell line, H89) (Niedz et al., 1995)Glycine max (soybean) (Plautz et al., 1996)Hordeum vulgar (barley) (Törmäkangas et al., 1995; Reichel et al., 1996)Nicotiana benthamiana & Nicotiana clevelandii (tobacco) (Baulcombeet al., 1995)Onion (Chiu et al., 1996)Tobacco (Beachy, 1995; Galbraith et al., 1995; Törmäkangas et al., 1995;Heinlein et al., 1995; Oparka et al., 1995; Chiu et al., 1996; Reichel et al.,1996)Zea mays (maize) (Galbraith et al., 1995; Hu & Cheng, 1995; Sheenet al., 1995; Chiu et al., 1996; Reichel et al., 1996)




B. Expression of GFP Fusion ProteinsGFP has been expressed as fusions to many proteins (Table II). In manycases, chimeric genes encoding either N- or C-terminal fusions to GFPretain the normal biological activity of the heterologous partner, as well asmaintaining fluorescent properties similar to native GFP (Flach et al., 1994;Wang & Hazelrigg, 1994; Marshall et al., 1995; Stearns, 1995). The use ofGFP and its variants in this capacity provides a “fluorescent tag” on theprotein, which allows for in vivo localization of the fusion protein. GFPfusions can provide enhanced sensitivity and resolution in comparison tostandard antibody staining techniques (Wang & Hazelrigg, 1994), and theGFP tag eliminates the need for fixation, cell permeabilization, and anti-body incubation steps normally required when using antibodies tagged withchemical fluorophores. Lastly, use of the GFP tag permits kinetic studies ofprotein localization and trafficking (Flach et al., 1994; Wang & Hazelrigg,1994). It is also possible to generate recombinant proteins fused to a bluevariant of GFP (such as P4-3; Rizzuto et al., 1996). CLONTECH offersseveral vectors designed to generate either C- or N-terminal fusions withEGFP (in all three reading frames), and C- and N-terminal fusion vectorsfor EBFP (Table I and Section III).

TABLE II. PROTEINS EXPRESSED AS FUSIONS TO GFP

Host Cell orProtein Organism Localization References

CotE Bacillus subtilis Forespore Webb et al., 1995

DacF, SpoIVA Bacillus subtilis Prespore/mother cell Lewis & Errington, 1996

Coronin D. discoideum Phagocytic cups Maniak et al., 1995

Myosin D. discoideum Moores et al., 1996

YopE cytotoxin Yersinia Jacobi et al., 1995

Histone H2B Yeast Nucleus Flach et al., 1994;Schlenstedt et al., 1995

Tubulin Yeast Microtubules Stearns, 1995

Nuf2p Yeast Spindle-pole body Silver, 1995; Kahana et al., 1995

Mitochondrial matrix Yeast Mitochondria Cox, 1995targeting signal

Npl3p Yeast Nucleus Corbett et al., 1995

Nucleoplasmin Yeast Nucleus Lim et al., 1995

Cap2p Yeast Waddle et al., 1996

Swi6p Yeast Sidorva et al., 1995

HMG-R Yeast Hampton et al., 1996

Bud10p Yeast Halme et al., 1996




TABLE II. PROTEINS EXPRESSED AS FUSIONS TO GFP continued

Host Cell orProtein Organism Localization References

Pmp47 Yeast Peroxisome Dyer et al., 1996

Act1p, Sac6p, Abp1p Yeast Cytoskeleton Doyle et al., 1996

p93dis1 Fission yeast Spindle-pole body &microtubules Nabeshima et al., 1995

Exu Drosophila Oocytes Wang & Hazelrigg, 1994

Mei-S332 Drosophila Centromere Kerrebrock et al., 1995

Tau Drosophila Microtubules Brand, 1995

β-galactosidase Drosophila cell movement Shiga et al., 1996

Streptavidin Insect cells Oker-Blom et al., 1996

ObTMV movementprotein Tobacco Filaments Beachy, 1995;

Heinlein et al., 1995

Potato virus Xcoat protein Potato plant Viral infection Santa Cruz et al., 1996

GST Zebrafish Cytoplasmic(?) Peters et al., 1995

11β-HSD2 Mammalian cells Endoplasmic reticulum Náray-Fejes-Tóth et al., 1996

MAP4 Mammalian cells Microtubules Olson et al., 1995

Mitochondrialtargeting signal Mammalian cells Mitochondria Rizzuto et al., 1995

Cyclins Mammalian cells Nucleus, micro-tubules, or vesicles Pines, 1995

PML Mammalian cells Ogawa et al., 1995

Human glucocor- Mammalian cells Ogawa et al., 1995ticoid receptor Carey et al., 1996

Rat glucocorticoid Mammalian cells Rizzuto et al., 1995receptor Htun et al., 1996

Chromogranin B HeLa cells Secreted Kaether et al., 1995

HIV p17 protein HeLa cells Nucleus Bian et al., 1995

NMDAR1 HEK293 cells Membrane Marshall et al., 1995

CENP-B Human cells Nucleus Sullivan et al., 1995



C. GFP Expression in Mammalian CellsAppropriate vectors may be transfected into mammalian cells by a varietyof techniques, including those using calcium phosphate (Chen & Okayama,1988), DEAE-dextran (Rosenthal, 1987), various liposome-based trans-fection reagents (Sambrook et al., 1989), and electroporation (Ausubel etal., 1994). Any calcium phosphate transfection procedure may be used, butwe recommend using the CalPhos MaximizerTM Transfection Kit (#K2050-1) for the highest calcium phosphate-mediated transfection efficiencieswith an incubation of only 2–3 hours. Likewise, any liposome-mediatedtransfection procedure may be used, but we recommend using CLONfectinTM

(#8020-1) for high, reproducible transfection efficiencies in a variety of celltypes. If you purchase CalPhos Maximizer or CLONfectin, follow thetransfection guidelines provided with those products. For further informa-tion on cell culture techniques, we recommend Culture of Animal Cells,Third Edition, R. I. Freshney (1993, Wiley-Liss; available from CLONTECHPress, #V2128-1).

The efficiency of a mammalian transfection procedure is primarily depen-dent on the host cell line. Different cell lines may vary by several orders ofmagnitude in their ability to take up and express exogenous DNA. More-over, a method that works well for one host cell line may be inferior foranother. Therefore, when working with a cell line for the first time, it isadvisable to compare the efficiencies of several transfection protocols. Thiscan best be accomplished using one of the GFP vectors which has the CMVimmediate early promoter for high-level expression in most cell lines. Thepromoterless pEGFP-1 or pGFP-1 vector may be used to determine thebackground autofluorescence in the host cell line. After a method oftransfection is chosen, it must be optimized for parameters such as celldensity, the amount and purity of DNA, media conditions, transfection time,and the posttransfection interval required for GFP detection. Once opti-mized, these parameters should be kept constant to obtain reproducibleresults.

After a calcium phosphate-based or liposome-mediated transfection pro-cedure, wait 24–72 hr before you assay for transient gene expression orstart selection for stable transformants. In most cases, GFP expressionmay be detected by fluorescence microscopy, FACS analysis, or fluoro-meter assays 24–72 hours after transfection, depending on the host cellline used. If you used electroporation, wait until 48 hr posttransfection toassay or begin selection. For visualizing GFP-expressing cells by fluores-cence microscopy, grow the cells on a sterile glass coverslip placed in a60-mm culture plate. Alternatively, an inverted fluorescence microscopemay be used for direct observation of fluorescing cells in the culture plate.We are aware of one report of a stable mammalian cell line expressing GFP(Olson et al., 1995).




D. GFP Expression in Plants

TABLE III. USE OF GFP IN PLANTS

Species (culture) Method Reference

Arabidopsis thaliana Agrobacterium (stable) Haseloff & Amos, 1995(thale cress roots) Particle bombardment Chiu et al., 1996

Citrus sinensis (L.) Osbeck cv. Hamlin Electroporation Niedz et al., 1995(embryogenic suspension culturesweet-orange cells H89)

Glycine max Particle bombardment Plautz et al., 1996(soybean suspension culture)

Hordeum vulgar Particle bombardment Törmäkangas et al., 1995(barley)

Nicotiana benthamiana PVX infection Baulcombe et al., 1995(tobacco plant leaves)

Nicotiana clevelandii PVX infection Baulcombe et al., 1995(tobacco plant leaves)

Onion epidermal cells Particle bombardment Chiu et al., 1996

family Solanaceae PVX infection Santa Cruz et al., 1996

Tobacco Particle bombardment Törmäkangas et al., 1995;Sheen et al., 1995

Electroporation or PEG Galbraith et al., 1995;Chiu et al., 1996

ObTMV infection Beachy, 1995;Heinlein et al., 1995

Zea mays Electroporation or PEG Hu & Cheng, 1995;(maize protoplast suspension culture) Electroporation Galbraith et al., 1995;

Chiu et al., 1996

GFP has been expressed in several plant species by a variety of methods(see Table III). GFP allows plant scientists to study transformed cells andtissues in vivo, without sacrificing vital cultures. The wt GFP codingsequences contain a region recognized in Arabidopsis as a cryptic plantintron (bases 400 and 483). The intron is efficiently spliced out in Arabidopsisresulting in a nonfunctional protein (Haseloff & Amos, 1995). Functionalwt GFP has been transiently expressed in several other plant speciesindicating that the cryptic intron may not be recognized or is recognized lessefficiently in other plant species. A modified version of wt GFP, in which thecryptic sequences have been altered, has been used for GFP expressionin Arabidopsis (Haseloff & Amos, 1995). The cryptic intron has also beenremoved by the changes to codon usage in hGFP-S65T and EGFP.The




same GFP gene as that contained in the phGFP-S65T Vector has beensuccessfully used to express GFP in many plant systems using a variety oftransformation techniques (Chiu et al., 1996). EGFP contains only oneadditional amino acid mutation (Phe-64 to Leu), but is ~7-fold brighter thanGFP-S65T; therefore, EGFP should also be a useful reporter in plantexpression systems.

E. GFP Expression in Yeastwt GFP and GFPuv both express well in yeast. However, for expression inyeast, the GFP or GFPuv gene contained in CLONTECH's vectors mustfirst be isolated (by PCR or cut out of the plasmid) and inserted into anappropriate yeast expression vector by any of several standard methods(Sambrook et al., 1989). Note that GFP genes with human codon usage(i.e., hGFP-S65T, EGFP, and EBFP) are not efficiently expressed in yeast(Cormack et al., 1997). Furthermore, S. cerevisiae strains carrying theade2 mitochondrial mutation accumulate a red pigment, which gives thecells an autofluorescence that interferes with detection of GFP (Smirnov etal., 1967; Weisman et al., 1987).

For additional information on yeast growth and maintenance, preparationof yeast competent cells, and transformation of yeast cells, please call ourLiterature Request Line at 800–662–2566(CLON) or 415–424–8222,extension 2, or contact your local distributor, and request a copy of theCLONTECH Yeast Protocols Handbook (PT3024-1). Additionally, werecommend Guide to Yeast Genetics and Molecular Biology, C. Guthrieand G. R. Fink eds. (Vol. 194 in Methods in Enzymology, 1991, AcademicPress; available from CLONTECH Press, #V2010-1).




A. Filter Setswt GFP absorbs UV and blue light with a maximum absorbance at 395 nmand a minor peak of absorbance at 470 nm. The filter sets commonly usedwith fluorescein isothiocyanate (FITC) and GFP illuminate at450–500 nm—well above the major excitation peak of wt GFP. These filtersets therefore produce a fluorescent signal from wt GFP that is several-foldless than would be obtained with filter sets that excite at 395 nm. However,use of the major peak at 395 nm for excitation of the wt GFP is not advisabledue to rapid photoisomerization and loss of the fluorescent signal. Forsystems in which standard UV light is used for the detection of fluores-cence, GFPuv should be used.

In contrast to wt GFP, EGFP has a single, strong, red-shifted excitationpeak at 488 nm, making it well suited for detection in commonly usedequipment which utilize FITC optics. Maximal emission is achieved whenEGFP is excited at 488 nm, which corresponds perfectly to the 488 nm lineof argon ion lasers used in many fluorescence-activated cell sorting (FACS)machines and confocal scanning laser microscopes. In fact, this variantwas selected specifically on the basis of its increased fluorescence in FACSassays. Filter sets commonly used in fluorescence microscopy or fluoro-metry illuminate at 450–500 nm; therefore, EGFP is ideal for use in thesesystems as well. The major excitation peak of both GFP-S65T and RSGFPalso encompasses the excitation wavelength commonly used for FACSanalysis, confocal scanning laser microscopes, fluorometry, and fluores-cence microscopy. In practical terms, this means the detection limits in bothmicroscopy and FACS should be considerably lower with EGFP, GFP-S65T, or RSGFP. Also, living cells and animals tolerate longer wavelengthlight better due to the lower energies; therefore, the fluorescent signalobtained with illumination of these variants at ~488 nm is more stable andless toxic than the fluorescence obtained with wt GFP excited at 395 nm.

Chroma Technology Corporation (Tel: 802-257-1800; Fax: 802-257-9400;72 Cotton Mill Hill, Unit A-9, Brattleboro, VT 05301) has developed severalfilter sets designed for use with GFP; they claim the High Q FITC filter set(#41001) produces the best signal-to-noise ratio for visual work, and theHigh Q GFP set (#41014) produces the strongest absolute signal, but withsome background. We have also used a Zeiss filter set (#487909) with a450–490 nm band-pass excitation filter, 510 nm dichroic reflector and520–750 nm long-pass emission filter, and the Chroma filter set #31001.Other filter sets may give better performance, and it is necessary to matchthe filter set to the application.

For detection of EBFP, we recommend a filter set with a 380±15-nmbandpass excitation filter, a 420-nm long-pass dichroic reflector, and a460±25-nm bandpass filter. DAPI filter sets are not recommended because

V. Detection of GFP and GFP Variants



V. Detection of GFP and GFP Variants continued

they have a broad excitation window that lets through shorter wavelengthUV, which results in very rapid photobleaching of EBFP. Again, Chromahas developed specific sets for blue variants of GFP. Sets used for P4 orP4-3 (Heim & Tsien, 1996) are suitable for EBFP.

B. Flow CytometrySeveral investigators have sorted GFP-expressing cells by flow cytometry(Cheng & Kain, 1995; Ropp et al., 1995; Yu & van den Engh, 1995). Inparticular, good results have been obtained in FACS analysis with plantprotoplasts, yeast, E. coli, mammalian cells, and mammalian cells infectedwith bacteria expressing GFP (Atkins & Izant, 1995; Fey et al., 1995; Sheenet al., 1995; Cheng et al., 1996). Note that flow cytometry of EBFP-expressing cells requires a light source such as a krypton laser that canprovide excitation in the UV range (Ropp et al., 1996).

C. Fluorescence Microscopy1. Materials required

• 95% Ethanol• Phosphate buffered saline (PBS; pH 7.4)

Final Conc. To prepare 2 L of solutionNa2HPO4 58 mM 16.5 gNaH2PO4 17 mM 4.1 gNaCl 68 mM 8.0 g

Dissolve components in 1.8 L of deionized H2O. Adjust to pH 7.4 with0.1 N NaOH. Add H2O to a final volume of 2 L. Store at room temperature.• PBS/4% paraformaldehyde (pH 7.4–7.6)

Add 4 g of paraformaldehyde to 100 ml of PBS. Heat to dissolve.Store at 4°C.

• Rubber cement or molten agarose2. Fluorescence microscopy procedure

In a tissue culture hood:a. Sterilize glass coverslip:

i. Place coverslip into 95% ethanol. Shake excess ethanol fromcoverslip until it is nearly dry.

ii. Flame-sterilize coverslip and allow to completely air-dry insterile tissue-culture dish.

b. Plate and transfect cells in tissue-culture dish containing coverslip(s).c. At the end of culture period, remove the tissue culture media and

wash cells 3 times with PBS.




d. For fixed cells:i. After cells have been washed with PBS, add 2.0 ml of freshly

made PBS/4% paraformaldehyde directly on coverslip.ii. Incubate cells in solution at room temperature for 30 min.iii. Wash cells twice with PBS.iv. Proceed with steps for unfixed cells.

e. For unfixed cells:i. Carefully remove the coverslip from the plate with forceps. (Pay

close attention to which side of the coverslip contains the cells.)ii. Mount the coverslip onto a glass microscope slide.

a) Place a tiny drop of PBS on the slide, and allow the coverslipto slowly contact the solution and lie down on the slide.

b) Carefully aspirate the excess solution around the edge of thecoverslip using a Pasteur pipette connected to a vacuum pump.

c) Attach coverslip to the microscope slides with either moltenagarose or rubber cement.Note: Do not use nail polish, as this reagent inhibits GFP fluorescence.

d) Allow to dry for 30 min.iii. Immediately examine slides by fluorescence microscopy, or

store them at 4°C for up to 2–3 weeks .




Figure 5. Comparison of fluorescence intensity of GFP in sonication buffer (S.B.) versus GFPin cell lysates. LB was inoculated with a culture of untransformed JM109 cells and incubatedovernight at 30°C. Cell lysates were prepared as described in the protocol. Two-fold serial dilutionsof rGFP ranging from 2.5 µg to 9.5 ng were prepared in both sonication buffer and JM109 cell lysate.Fluorescence intensity was measured in a modified Hoefer Pharmacia Biotech, Inc. DyNA QuantTM

200 Fluorometer using an excitation filter of 365 nm and an emission filter of 510 nm. Similar resultscan also be obtained using rGFP-S65T. (RFU = relative fluorescence units.)

D. Quantitative Fluorometric AssayGFP can also be used in fluorometric assays to confirm and measure GFPfluorescence. Furthermore, using known amounts of purified recombinantGFP or GFP variant (rEGFP, rGFP-S65T, rGFPuv, or rGFP), fluorometricassays can be used to quantify the expression of GFP or GFP variant. Aftergenerating a standard curve using known amounts of recombinant protein,the fluorescence intensity of experimental samples can be measured andcompared to the standard curve to determine the amount of GFP expressedin the sample. A detailed method for quantitation of wt GFP levels using theTD-700 fluorometer is available from Turner Designs, Inc. (408-749-0994;http://www.turnerdesigns.com). When generating the standard curve, therecombinant protein should be diluted in the same buffer as the experimen-tal samples. In some expression systems, GFP can be detected directly inthe fluorometer; in other cases, cells will need to be disrupted in order tooptimally detect GFP fluorescence. Figure 5 shows a comparison of thefluorescence intensity of GFP detected in sonication buffer and in a clarifiedbacterial cell lysate. These results illustrate that the sensitivity and thelinear range of detection of GFP are relatively unaffected by the cell lysatebackground.

100

1

2

3

4

2

Log GFP (ng)

GFP in 6.25 µg JM109 lysateGFP in S.B.

Log

RFU

3 4




The following procedures have been developed using CLONTECH’sRecombinant GFP Protein (rGFP; see Section VI; #8360-1, -2). Use anexcitation filter of 365 nm and an emission filter of 510 nm when assayingwt GFP or GFPuv, and an excitation filter of 450–490 nm and an emissionfilter of 510 nm when assaying EGFP and GFP-S65T.Note: “rGFP” refers to recombinant wt GFP or one of the recombinant GFP variants.

1. Materials required• Sonication buffer (pH 8.0)

50 mM NaH2PO410 mM Tris-HCl (pH 8.0)

200 mM NaClAdjust pH to 8.0. Store at room temperature.

• PBS (pH 7.4; see Section V.C.1)2. Generating a GFP standard curve:

a. Prepare serial dilutions of rGFP in sonication buffer, deionized H2O,or 10 mM Tris-HCl (pH 8.0).Note: Samples can be stored at –20°C.

b. Assay dilutions in a fluorometer.c. Plot the log of the relative fluorescence units (RFU) as a function of

the log of fold-dilution of each sample such as the standard curveshown in Figure 6.

3. Measuring fluorescence intensity of bacterial samples:a. Pellet 20 ml of bacterial cell culture.b. Remove the supernatant and freeze pellet at –70°C.c. Wash pellet once with 1.6 ml of PBS.d. Resuspend pellet in 1.6 ml of sonication buffer.e. Prepare 2-fold serial dilutions in sonication buffer.f. Assay dilutions in a fluorometer.g. Compare results with those of the standard curve to determine the

amount of GFP protein expressed in the experimental samples.4. Preparation of cell lysates:

a. Pellet 100 ml of bacterial cell culture and remove the supernatant.Note: Cell pellets may be stored at –70°C for later use.

b. Wash pellet twice with 4 ml of PBS.c. Resuspend pellet in 4 ml of sonication buffer.d. Freeze/thaw sample in a dry ice/ethanol bath five times.e. Vortex sample for 1 min, and then incubate on ice for 1 min.



f. Repeat Step e two more times.g. Run sample through an 18-gauge needle several times.h. Transfer cell lysate to a 1.5-ml microcentrigue tube and centrifuge

at 12,000 rpm for 5 min at 4°C.i. Collect the supernatant and determine the protein concentration

using a standard assay (e. g., Bradford, Lowry, etc.; Scopes, 1987).j. Assay the dilutions in a fluorometer.

k. Compare the results with the standard curve to determine theamount of GFP expressed in the experimental samples.


Figure 6. Relative fluorescence intensity of two-fold serial dilutions of a suspension of GFP-transformed cells in sonication buffer. LB was inoculated with JM109 cells transformed with aplasmid encoding wt GFP and incubated overnight at 30°C. Fluorescence intensity was measuredby reading 5-µl samples of 2-fold dilutions in a modified Hoefer Pharmacia Biotech, Inc. DyNA Quant200 Fluorometer using an excitation filter of 365 nm and an emission filter of 510 nm.

00

1

2

1

Log Fold Dilution

Log

RFU

2



A. General InformationCLONTECH’s Recombinant Green Fluorescent Protein (rGFP; #8360-1),Recombinant GFP-S65T (rGFP-S65T; #8364-1), Recombinant EGFP(rEGFP; #8365-1), and Recombinant GFPuv (rGFPuv; #8366-1) are puri-fied from transformed E. coli using a method which ensures optimal purityof the recombinant protein and maintenance of GFP fluorescence. Allproteins are 27-kDa monomers with 239 amino acids. The excitation andfluorescence emission spectra for the rGFP is identical to GFP purified fromAequorea victoria (Chalfie et al., 1994). The purified proteins retain theirfluorescence capability under many harsh conditions (Section II.E) and aresuitable as control reagents for GFP expression studies using the LivingColors line of GFP reporter vectors.

Applications of purified rGFP and rGFP variant proteins include use asstandards for SDS or two-dimensional PAGE, isoelectric focusing, West-ern blot analysis, calibration of fluorometers and FACS machines, fluores-cence microscopy, and microinjection of GFP into cells and tissues.

B. rGFP and rGFP Variants as Standards in Western BlotsWhen used as a standard for Western blotting applications in conjunctionwith GFP Monoclonal Antibody (#8362-1) or GFP Polyclonal Antibody (IgGFraction; #8363-1), the recombinant proteins can be used to correlate GFPexpression levels to fluorescence intensity or to differentiate problems withdetection of GFP fluorescence from expression of GFP protein.

Use a standard procedure for discontinuous polyacrylamide gel electro-phoresis (PAGE) to resolve the proteins on a one-dimensional gel (Laemmli,1970). If another electrophoresis system is employed, the sample prepa-ration should be modified accordingly. Just prior to use, allow rGFP proteinto thaw at room temperature, mix gently until solution is clear, and thenplace tube on ice.

On a minigel apparatus, 25–75 µg of lysate protein per lane is typicallyneeded for satisfactory separation (i.e., discrete banding throughout themolecular weight range) of a protein mixture derived from a whole cell ortissue homogenate. If rGFP is to be used as an internal standard in aCoomassie blue-stained minigel, we recommend loading 500 ng of rGFPper lane. If rGFP is added to a total cell/tissue lysate or other crude sample,the amount of total protein loaded per lane must be optimized for theparticular application.

For Western blotting applications, we recommend loading 40–400 pg ofrGFP (or rGFP variant) per lane for a strong positive signal usingCLONTECH’s GFP Monoclonal or Polyclonal Antibody in conjunction witha chemiluminescent detection system (Kain et al., 1994). Typically, the

VI. Purified Recombinant GFP and GFP Variants



GFP Monoclonal and Polyclonal Antibodies can be used at 1:500 and1:1000 dilutions, respectively. Please refer to Section VII and the ProductAnalysis Certificate (PAC) provided with each antibody for further informa-tion on using CLONTECH's GFP Antibodies.

Some GFP fluorescence may be observed when the protein is resolved onan SDS-PAGE gel if nanogram quantities of rGFP are present. Generally,rGFP will not fluoresce on a Western blot.

VI. Purified Recombinant GFP and GFP Variants continued



C. rGFP and rGFP Variants as a Control for Fluorescence MicroscopyThe following two protocols are for use of rGFP or rGFP variants as a controlon microscope slides in fluorescence microscopy. The purified proteinsmay be used to optimize lamp and filter set conditions for detection of GFPfluorescence, or as a qualitative means to correlate GFP fluorescence withthe amount of protein in transfected cells.

1. Unfixed samplesUse this method for live cell fluorescence or other cases where afixation step is not desired.a. Perform 1:10 serial dilutions of the 1.0 mg/ml rGFP or rGFP variant

stock solution with 10 mM Tris-HCl (pH 8.0) to yield concentrationsof 0.1 mg/ml and 0.01 mg/ml.Notes:

• These dilutions should suffice as a positive control. The 1.0 mg/ml solution willgive a very bright fluorescent signal by microscopy.

• The diluted samples can be aliquoted and stored frozen at –70°C for up to1 yr with no loss of fluorescence intensity. Avoid repeated freeze-thaw cycleswith the same aliquot.

b. Using a micropipette, spot 1–2 µl of diluted protein onto themicroscope slide. If using a slide that contains a mounted coverslip,position the spot several millimeters away from the sample suchthat a second coverslip can be added over the protein spot.

c. Allow the protein to air-dry for a few seconds, and mark the positionof the spot on the other side of the slide to aid in focusing.

d. Add a coverslip over the spot using a 90% glycerol solution in100 mM Tris-HCl (pH 7.5).

e. Fluorescence from the spot is best viewed at low magnification,using either a 10X or 20X objective.

2. Fixed samplesIn some cases it may be necessary to fix the recombinant protein to themicroscope slide prior to microscopy. This can be done by dipping thesection of the microscope slide containing the air-dried protein spot(after Step 1.c above) into 100% methanol for 1 min. Allow the slide todry completely and place a coverslip over the sample as in Step 1.dabove.

VI. Purified Recombinant GFP and GFP Variants continued



A. Applications for GFP AntibodiesCLONTECH offers two antibodies, GFP Monoclonal Antibody (#8362-1)and GFP Polyclonal Antibody (IgG Fraction; #8363-1, -2). Both antibodiesspecifically recognize wt GFP, each of the red-shifted GFP variants, EBFP,and GFPuv. GFP Monoclonal Antibody is purified from the serum-freemedia of mouse hybridoma cultures. Since the GFP Monoclonal Antibodyonly recognizes a single epitope of GFP, the signals are highly specific andhave minimal background. GFP Polyclonal Antibody is a purified fraction ofan Anti-rGFP Antiserum that has been purified by passage through aprotein G column to remove nonspecific immunoglobulins from the serum.

Both antibodies can be used to confirm GFP protein expression by Westernblot analysis and to correlate levels of GFP protein expression withfluorescence intensity (detected with microscopy, flow cytometry, or fluo-rometry). Typically, the GFP Monoclonal and Polyclonal Antibodies can beused at 1:500 and 1:1000 dilutions, respectively, for Western blottingapplications. Please refer to the Product Analysis Certificate (PAC) en-closed with each antibody for recommended dilutions. Avoid repeatedlyfreezing and thawing the GFP Antibodies.

In addition to detection of GFP and its variants on Western blots, the GFPantibodies can be used to immunoprecipitate GFP-fusion proteins (Silver,pers. comm.) and for in situ detection of GFP (Lauderdale, pers. comm.).An immunoprecipitation procedure, modified for use with GFP MonoclonalAntibody, is provided below. This procedure has been optimized usingmammalian cells expressing GFP, but should work with lysates preparedfrom any cell type. However, if you are using a bacterial or yeast host, youwill need to perform additional steps to break down the cell wall.

B. Immunoprecipitation with GFP Monoclonal Antibody1. Solutions Required

• Lysis buffer (100 mM Potassium Phosphate [pH 7.8] + 0.2% TritonX-100) To make 10 ml of Lysis Buffer, mix together:

9.15 ml of 100 mM K 2HPO40.85 ml of 100 mM KH2PO420 µl of Triton X-100 (0.2% final)

If necessary, adjust pH to 7.8. Store buffer at room temperature.Just prior to use, add 10 µl of a 1 M stock solution of DTT (1 mM finalconcentration) and an appropriate protease inhibitor or combina-tion of inhibitors, such as the Protease Inhibitor cocktail fromBoehringer Mannheim (#1697 498).

VII. GFP Antibodies



• 1.0 M DithiothreitolAdd 1.54 g of dithiothreitol to 8 ml of deionized H2O and mixgently until dissolved. Adjust final volume to 10 ml with deionizedH2O. Aliquot into microcentrifuge tubes and freeze at –20°C.

• GFP Monoclonal Antibody , diluted to 1 mg/ml in PBS (pH ~7).• Protein A Sepharose beads,

Spin down beads to remove the ethanol; then wash beads oncein PBS and resuspend them at 1 mg/ml in PBS.

• PBS (see Section IV.C.1 for recipe)

2. Procedurea. Spin down ~ 1 x 106 cells and resuspend the cell pellet in 20 ml of

cold (4°C) Lysis Buffer containing 1 mM DTT and protease inhibi-tors.

b. Place cells on ice for 45 min.c. Clear the lysate by pelleting the cells at 12,000 x g for 10 min at 4°C.d. Transfer 500 µl of cleared lysate to a 1.5-ml microcentrifuge tube.e. Add the diluted mAb to a final concentration of 2–10 µg/ml.f. Place the tube at 4°C for 2 hr with continuous gentle inversion.g. Add 40 µl of Protein A Sepharose beads to the antibody-antigen

mixture in a 1:1 suspension in PBS.h. Vortex and incubate at 4°C for 1 hr with continuous gentle inversion.i. Centrifuge at 10,000 x g for 10 min at 4°C.

3. Analysis of samplea. Wash the beads with 1 ml of Lysis Buffer.b. Repeat this wash procedure twice.c. Add 50 µl of 1X SDS-PAGE sample buffer to beads.d. Vortex, then boil for 10 min.e. Centrifuge at 12,000 x g for 5 min at room temperature.f. Electrophorese the supernatant on a polyacrylamide gel and ana-

lyze the resolved proteins on a Western blot.Notes:

• Load 10–20 µl of supernatant per well (mini-gel)

• We recommend the Western ExposureTM Chemiluminescent Detection System(#K2030-1, #K2031-1) for detection of positive signals.

VII. GFP Antibodies continued



VIII. Troubleshooting Guide

A. Potential Difficulties in Using GFP Fluorescence• Variability in the intensity of GFP fluorescence has been noted. This

may be due in part to the relatively slow formation of the GFPchromophore and the requirement for molecular oxygen (Heim et al.,1994).

• The slow rate of chromophore formation and the apparent stability ofwt GFP may preclude the use of GFP as a reporter to monitor fastchanges in promoter activity (Heim et al., 1994, Davis et al., 1995). Thislimitation is reduced by use of EGFP or GFP-S65T, which acquirefluorescence faster than wt GFP (Heim et al., 1994).

• The wt GFP coding sequences contain a cryptic plant intron (betweenbases 400 and 483) that is efficiently spliced out in Arabidopsis andresults in a nonfunctional protein (Haseloff & Amos, 1995). Functionalwt GFP has been transiently expressed in several other plant speciesindicating that the cryptic intron may not be recognized or recognizedless efficiently in these species. hGFP-S65T and EGFP do not containthe cryptic intron and can be used for expression in plants.

• Some people have put GFP expression constructs into their systemand failed to detect fluorescence. There can be numerous reasons forfailure, including use of an inappropriate filter set, expression of GFPbelow the limit of detection, and failure of GFP to form the chro-mophore. Recombinant GFP Protein and GFP Monoclonal or PolyclonalAntibody may be used to troubleshoot GFP expression in these cases.Since GFPuv and EGFP fluoresce brighter than wt GFP, they are betteralternatives for expression of GFP.

• GFP targeted to a low pH environment may lose fluorescence (seeSection II.E.4).

B. Requirements for GFP Chromophore Formation• Formation of the chromophore in wt GFP and GFP-S65T appears to be

temperature sensitive; however, the mutations in EGFP and GFPuvsuppress this thermosensitivity (see Section II.E.7). In some cases,E. coli, yeast, and mammalian cells expressing wt GFP or GFP-S65Thave shown stronger fluorescence when grown at lower temperatures(Heim et al., 1994; Ward, pers. comm.; Lim et al., 1995; Pines, 1995;Ogawa et al., 1995). Hence, incubation at a lower temperature mayincrease the fluorescence signal obtained when using wt GFP andGFP-S65T.

• GFP chromophore formation requires molecular oxygen (Heim et al.,1994; Davis, D. F. et al., 1995); therefore, cells must be grown underaerobic conditions.



VIII. Troubleshooting Guide continued

C. Photobleaching or Photodestruction of Chromophore• Excite at 470 nm for wt GFP, 360–400 nm for GFPuv, 360–400 nm for

EBFP, and 488 nm for the red-shifted GFP variants. Excitation at the395-nm peak for wt GFP may result in rapid loss of signal. For GFPuvand EBFP, use the longest possible excitation wavelength to minimizethe rate of photobleaching.

• A tungsten-QTH or argon light source is preferable. Mercury and xenonlamps produce significant UV radiation which will rapidly destroy thechromophore unless strongly blocked by appropriate filters.

D. Autofluorescence• Some samples may have a significant background autofluorescence,

e.g., worm guts (Chalfie et al., 1994; Niswender et al., 1995). Abandpass emission filter may make the autofluorescence appear thesame color as GFP; using a long-pass emission filter may allow thecolor of the GFP and autofluorescence to be distinguished. Use ofDAPI filters may also allow autofluorescence to be distinguished(Brand,1995; Pines, 1995).

• Most autofluorescence in mammalian cells is due to flavin coenzymes(FAD and FMN; Aubin, 1979) which have absorption and emissionmaxima at 450 and 515 nm respectively. These values are very similarto those for wt GFP and the red-shifted GFP variants, soautofluorescence may obscure the GFP signal. The use of DAPI filterswhen using 450/515 nm light for excitation may make autofluorescenceappear blue while the GFP signal remains green. In addition, somegrowth media can cause autofluorescence. Since GFPuv uses excita-tion of 360–400 nm, autofluorescence of flavins should not be aproblem. When possible, perform microscopy in a clear buffer such asPBS, or medium lacking phenol red.

• Some cell types can produce a speckled autofluorescence patternwhich is likely due to mitochondrially bound NADH (Aubin, 1979). Thisproblem is minimized with excitatory light around 488 nm, as opposedto UV excitation (Niswender et al., 1995). Therefore, we recommendusing EGFP for expression in these systems. Such problems areunavoidable with GFPuv and EBFP, whose excitation requires light inthe UV range (360–400 nm).

• For mammalian cells, autofluorescence can increase with time inculture. For example, when CHO or SCI cells were removed fromfrozen stocks and reintroduced into culture, the observed autofluores-cence (emission at 520 nm) increased with time until a plateau wasreached around 48 hours (Aubin, 1979). Therefore, in some cases itmay be preferable to work with freshly plated cells.




• Always use a mock-transfected control or cells transfected with apromoterless vector such as pEGFP-1 to gauge the extent ofautofluorescence.

• For fixed cells, autofluorescence can be reduced by washing with0.1% sodium borohydride in PBS for 30 min after fixation.

E. Considerations for Mammalian Expression• If you have not been able to detect wt GFP in a mammalian expression

system, try using EGFP or EBFP instead. The brighter fluorescenceand improved translation should make detection of the expression ofthese variants easier than expression of wt GFP.

• Have you verified your GFP plasmid construct and concentration witha restriction digest? Verify that all subcloning steps have been donecorrectly, keeping in mind specific restriction sites in some vectorswhich are inactivated by methylation. CLONTECH’s GFP and EGFPSequencing Primers may be used to verify sequence junctions.

• Is the vector compatible with your cell type? Has the CMV promoterbeen used previously for transient expression in your cells?

• Do you have another assay to estimate transfection efficiency? Thiscan be accomplished by transfection with a second reporter plasmidwhich contains the same promoter element, like pCMVβ (#6177-1),and employing standard assays or stains to detect β-galactosidaseactivity. Alternatively, use a secreted reporter such as secreted alkalinephosphatase (SEAP), which can be quantified using the culture me-dium, leaving the cells intact for analysis of GFP expression.

• Expression of GFP protein can be verified by Western analysis usingeither the GFP Monoclonal or Polyclonal Antibody.

F. Optimizing Microscope/FACS Applications• In general, optimal visual detection of GFP fluorescence by microscopy

is achieved in a darkened room, after your eyes have adapted for10–20 minutes.

• In microscopy, the primary issue is the choice of filter sets. Choose thefilter set that is optimal for the GFP variant that you are using. Ingeneral, conditions used for fluorescein should give some signal withall variants except GFPuv. Autofluorescence may be a problem insome cell types or organisms. For more information on the filter setsrecommended for GFP and its variants, see Section V.A.

• A simple control for the microscope setup is to spot a small volume ofpurified recombinant GFP protein (e.g., rGFP, rGFP-S65T, rEGFP, orrGFPuv) on a microscope slide. As a crude substitute, any source offluorescein can be used, such as a fluorescein-conjugated antibody oravidin-fluorescein.



• GFP fluorescence is very sensitive to some nail polishes used to sealcoverslips (Chalfie et al., 1994; Wang & Hazelrigg, 1994). In place ofnail polish for mounting coverslips, we recommend molten agarose orrubber cement.

• Exciting GFP intensely for extended periods may generate free radi-cals that are toxic to the cell. This problem can be minimized byexcitation at 450–490 nm.

G. Anomalous Band in some GFP Plasmid PreparationsWe have occasionally observed a band running at approximately 500 bp insome (not all) plasmid preparations of the GFP vectors that have thebackbone carrying the kanamycin resistance gene and the f1 origin (i.e.,pEGFP-1, pEGFP-N1, -N2, -N3, -C1, -C2, -C3, and pEBFP-N1, -C1). Thepresence and level of this band varies greatly from one plasmid DNApreparation to another. We have not thoroughly characterized this band;however, it appears to be a small circular DNA, possibly single-stranded,that is generated as a by-product of plasmid replication. As far as we canascertain, this band does not interfere with ligating inserts into the vectors,does not affect transformation or transfection efficiencies, and does nothave any deleterious effect on mammalian cells.




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X. Summary Reference List

BackgroundBrand, A. (1995)Chalfie, M., et al. (1994)Haseloff, J. & Amos, B. (1995)Hassler, S. (1995)Hodgkinson, S. (1995)Kolberg, R. (1994)Morin, J. G. & Hastings, J. W. (1971)Morise, H., et al. (1974)Pines, J. (1995)Prasher, D. C., et al. (1992)Prasher, D.C. (1995)Shimomura, O., et al. (1962)

Reviews on GFP technologyChalfie, M. (1995)Coghlan, A. (1995)Coxon, A. & Bestor, T. H. (1995)Cubitt, A. B., et al. (1995)Hassler, S. (1995)Kolberg, R. (1994)Prasher, D.C. (1995)Stearns, T. (1995)Youvan, D. C. (1995)

Properties of GFPBokman, S. H. & Ward, W. W. (1981)Chattoraj, M. et al. (1996)Cody, C. W., et al. (1993)Davis, D. F., et al. (1995)Inouye, S. & Tsuji, F. I. (1994)Niswender, K. D., et al. (1995)Niwa, H., et al. (1996)Ormö, M. et al. (1996)Robart, F. D. & Ward, W. W. (1990)Roth, A. (1985)Surpin, M. A. & Ward, W. W. (1989)Ward, W. W. (1979)Ward, W. W., et al. (1980)Ward, W. W. (1981)Ward, W. W. & Bokman, S. H. (1982)Yang, F., et al. (1996)

Mutagenesis of GFPCormack, B., et al. (1996)Crameri, A., et al. (1996)Delagrave, S., et al. (1995)Ehrig, T., et al. (1995)Heim, R., et al. (1994)Heim, R., et al. (1995)Heim, R. & Tsien, R. Y. (1996)Reichel, C., et al. (1996)Siemering, K. R., et al. (1996)Yang, T. T., et al.(1996a)

Expression of GFP in ProkaryotesBurlage, R., et al. (1995)Chalfie, M., et al. (1994)Crameri, A., et al. (1996)Dhandayuthapani, S. et al. (1995)Inouye, S. & Tsuji, F. I. (1994a)Inouye, S. & Tsuji, F. I. (1994b)Kahana, J. A., et al. (1995)Kain, S. R., et al. (1995)Kitts, P., et al. (1995)Kremer, L. et al. (1995)Yu, J. & van den Engh, G. (1995)

Expression of GFP in EukaryotesAtkins, D. & Izantz, J. G. (1995)Baulcombe, D. C., et al. (1995)Bonner, J. T., et al. (1995)Brand, A. (1995)Chalfie, M., et al. (1994)Chao, Y.-C., et al. (1996)Chiu, W.-L., et al. (1996)Davis, I., et al. (1995)Galbraith, D. W., et al. (1995)Halme, A., et al. (1996)Haselhoff, J. & Amos, B. (1995)Higgs, S., et al. (1996)Hodgkinson, S. (1995)Kahana, J. A. et al. (1995)Kain, S. R., et al. (1995)Kitts, P., et al. (1995)Lo, D. C., et al. (1994)Moss, J. B. & Rosenthal, N. (1994)Murphy, S. M. & Stearns, T. (1996)Niedz, R. P., et al. (1995)Ogawa, H., et al. (1995)Pines, J. (1995)Plautz, J. D., et al. (1996)Riles, L., et al. (1995)Schlenstedt, G., et al. (1995)Tannahill, D., et al. (1995)Törmäkangas, K., et al. (1995)Troemel, E. R., et al. (1995)Waddle, J. A., et al. (1996)Wu, G.-I., et al. (1995)Yang, T. T., et al. (1996a, b)Yu, K. L. & Dong, K. W. (1995)

Transgenic expression of GFPAmsterdam, A., et al. (1995) [Zebrafish]Barthmaier, P. & Fyrberg, E. (1995) [Drosophila]Chalfie, M., et al. (1994) [C. elegans]Chiu, W.-L., et al. (1996) [Tobacco]Davis, I. et al. (1995) [Drosophila]Haseloff, J. & Amos, B. (1995) [Arabidopsis]Higgs, S. et al. (1996) [Mosquito]Ikawa, M., et al. (1995a & 1995b) [Mouse]



X. Summary Reference List continued

Reichel, C. et al. (1996) [Various plants]Santa Cruz, S. et al. (1996) [Potato plant]Shiga, Y. et al. (1996) [Drosophila]Wang, S. & Hazelrigg, T. (1994) [Drosophila]Yeh, E. et al. (1995) [Drosophila]

Expression of GFP-fusion proteinsBian, J., et al. (1995)Carey, K. L. et al. (1996)Corbett, A. H., et al. (1995)Doyle, T. & Botstein, D. (1996)Dyer, J. M. et al. (1996)Flach, J., et al. (1994)Halme, A., et al. (1996)Hampton, R. Y., et al. (1996)Heinlein, M., et al. (1995)Htun, H. et al. (1996)Jacobi, C. A., et al. (1995)Kaether, C. & Gerdes, H.-H.(1995)Kahana, J. A., et al. (1995)Kerrebrock, A. W., et al. (1995)Lewis, P. J. & Errington, J. (1996)Lim, C. R., et al. (1995)Maniak, M., et al. (1995)Marshall, J., et al. (1995)Moores, S. L., et al. (1996)Nabeshima, K., et al. (1995)Náray-Fejes-Tóth, A. & Fejes-Toth, G. (1996)

Ogawa, H., et al. (1995)Oker-Blom, C. et al. (1996)Olson, K. R. et al. (1995)Oparka, K. J., et al. (1995)Peters, K. G., et al. (1995)Rizzuto, R., et al. (1995)Rizzuto, R., et al. (1996)Rutter, G. A. et al. (1996)Sengupta, P., et al. (1994)Sidorova, J. M., et al. (1995)Stearns, T. (1995)Sullivan, K. F. et al. (1995)Treinin, M. & Chalfie, M. (1995)Venerando, R., et al. (1996)Waddle, J. A., et al. (1996)Wang, S. & Hazelrigg, T. (1994)Webb, C. D., et al. (1995)

Retroviral expression of GFPCheng, L., et al. (1996)Levy, J. P., et al. (1996)Muldoon, R. R., et al. (1997)

Flow cytometry of GFP-expressing cellsAtkins, D. & Izant, J. G. (1995)Cheng, L. & Kain, S. (1995)Cheng, L., et al. (1996)Cormack, B. P., et al. (1996)Fey, P., et al. (1995)Galbraith, D. W., et al. (1995)Levy, J. P., et al. (1996)Ropp, J. D., et al. (1995)Ropp, J. D., et al. (1996)Sheen, J., et al. (1995)Subramanian, S. & Srienc, F. (1996)



XI. Related Products

Product Cat. #

Wild-Type GFP Vectors

• pGFP Vector 6097-1

• p6xHis-GFP Vector 6087-1

GFP Variant Vectors

• pGFPuv Vector 6079-1

• pBAD-GFPuv Vector 6078-1

• pEGFP Vector 6077-1

• pEGFP-1 Vector 6086-1

• pEGFP-N1 Vector 6085-1



• pEGFP-C1 Vector 6084-1



• phGFP-S65T Vector 6088-1

• pEBFP Vector 6068-1

• pEBFP-N1 Vector 6069-1

• pEBFP-C1 Vector 6070-1

• pIRES-EGFP Vector 6064-1

Other Products

• GFP-N Sequencing Primer 6476-1

• GFP-C Sequencing Primer 6477-1

• EGFP-N Sequencing Primer 6479-1

• EGFP-C Sequencing Primer 6478-1

• Recombinant GFP Protein 8360-1, -2

• Recombinant GFP-S65T Protein 8364-1, -2

• Recombinant EGFP Protein 8365-1

• Recombinant GFPuv Protein 8366-1

• GFP Monoclonal Antibody 8362-1



Product Cat. #

• GFP Polyclonal Antibody (IgG Fraction) 8363-1, -2

• CalPhos Maximizer™ 8021-1, -2

• CalPhos MaximizerTM Transfection Kit K2050-1

• CLONfectinTM 8020-1

• Great EscAPeTM SEAP Reporter System K2040-1

• Great EscAPeTM SEAP Detection Kit K2041-1

• pCMVβ Reporter Vector 6177-1

• Guide to Yeast Genetics and Molecular Biology V2010-1(Guthrie, C. & Fink, G. R., eds.)

• Culture of Animal Cells, Third Edition V2128-1(Freshney, R. I.)

• Western ExposureTM Chemiluminescent Detection Systemwith membranes K2030-1without membranes K2031-1

XI. Related Products continued



Appendix. Fluorescent Proteins Newsgroup

A newsgroup for the discussion of fluorescent proteins has been created withinthe bionet hierarchy of Newsgroups. This newsgroup is intended to provide aforum for discussion of bioluminescence, to promote further development ofreporter proteins obtained from bioluminescent organisms (e.g., GFP, luciferase,and Aequorin), and to facilitate their application to interesting biological ques-tions. (A full copy of the newsgroup charter can be found in the BIOSCI archives.)

We hope you will find this newsgroup useful and encourage you to participate inthe discussions.

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Administration: BIOSCI International Newsgroups for Biology

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Documents

Living Colors User Manualcoli.usal.es/web/abydl/biblioteca/bibelectro.alu/... · 1999. 2. 10. · B. Expresion of GFP Fusion Proteins 18 C. GFP Expression in Mammalian Cells 20 D