NOTES & TIPS174

6. Araki, N., Bobinson, D., and Nishimoto, S. K. (1993) J. Bonerat and mouse DNA as internal standards, respec-Mineral Res. 8, 313–322.tively. In the mouse DNA assay, a serial dilution of

7. Nudel, U., Zakut, R., Shani, M., Neuman, S., Lewy, Z., and Yaffe,mouse DNA (0.03–3.5 pM) was amplified with 0.7 pMD. (1983) Nucleic Acids Res. 11, 1759–1771.rat DNA, and, in the rat DNA assay, rat DNA (0.11–14

8. Alonso, S., Minty, A., Bourlet, Y., and Buckingham, M. (1986)pM) was amplified with 0.14 pM mouse DNA. A linearJ. Mol. Evol. 23, 11–22.

relationship of mouse/rat ratio to the various amounts9. Schwartz, H. E., and Ulfelder K. J. (1992) Anal. Chem. 64, 1737–of mouse DNA added was obtained in the mouse b- 1740.

actin assay (Fig. 2A). A similar linearity was observedin the rat DNA assay in which various amounts of ratDNA were amplified against a constant amount ofmouse DNA (Fig. 2B). Although the highly homologousmouse and rat b-actin DNA were amplified with thesame primers, the efficiency of amplification of rat DNA Spectrophotometric Determination of Iodixanolwas lower than that in mouse. There are 23 (out of 542

in Subcellular Fractions of Mammalian Cellsbp) different base pairs between mouse and rat b-actinfragments, and the number of G/C in the rat sequenceis slightly larger than that in mouse (13 G/C in rats

Martin Schroder, Rolf Schafer,1 and Peter Friedl2and 9 G/C in mouse), but the 7 bp at the antisense sideare all G/C in rats while all A/T in mouse. It is possible Technische Hochschule Darmstadt, Institut fur Biochemie,

Petersenstrasse 22, D-64287 Darmstadt, Germanythat this difference in the G/C location in the rat se-quence may cause the lower efficiency of amplificationcompared to mouse. Intraassay coefficients of variation

Received July 29, 1996of mouse and rat b-actin assays were 13.6 and 15.7%determined at the levels of mRNA collected from mouseand rat brains, respectively. When DNA samples syn- Nonionic iodinated density gradient media arethesized from mouse and rat brain mRNA samples widely used for the separation of cells and organelleswere serially diluted and were applied to each competi- (1). The most suitable of them is iodixanol. It displaystive PCR system, parallelism with the standard curve no cytotoxicity and a number of marker enzymes forof each assay system was observed (Fig. 2), indicating cellular organelles can be assayed in the presence ofthat mouse and rat b-actin could be measured by these iodixanol (2). In contrast to other nonionic iodinatedcompetitive PCR system. density gradient media, such as metrizamide or nyco-

Since the measurement of the expression of b-actin denz, iodixanol readily forms self-generating gradients,mRNA has been used for the standardization of the thus obliviating the preparation of gradients using aexpression of target mRNAs among samples, the estab- gradient marker.lishment of a sensitive b-actin mRNA assay system A subcellular fractionation of cellular organelles iswould be useful. Furthermore, the usage of an interspe- evaluated by the distribution of marker enzymes in thecies gene as the internal standard for each other would gradient and the density profile of the gradient. Thesimplify the construction of internal standards and per- density profile of a gradient is commonly analyzed bymit two assay systems to be performed simultaneously. measuring the refractive index of the gradient frac-

tions. Cellular material present in subcellular fractionsAcknowledgments. We thank Dr. R. L. Hawkins and Mr. C. Okerefor careful review and helpful advice on the manuscript. This study interferes with the determination of the refractive in-was supported by the Research Development Corporation of Japan. dex, thus necessitating a mock fractionation to deter-

mine the density profile of the gradient.REFERENCES Here we report the direct and specific determination

of iodixanol as an example for a nonionic density gradi-1. Gilliland, G., Perrin, S., and Bunn, H. F. (1990) in PCR Protocols:A Guide to Methods and Applications (Innis, M. A., Gelfand, ent medium in subcellular fractions. No mock runs areD. H., Sninsky, J. J., and White, T. J., Eds.), Vol. 1, pp. 60–69, necessary to determine the density profile of the gradi-Academic Press, San Diego. ent when this method is used. This assay should be

2. Gilliland, G., Perrin, S., Blanchard, K., and Bunn, H. F. (1990) more convenient for many laboratories because a re-Proc. Natl. Acad. Sci. USA 87, 2725–2729.fractometer is not required.3. Wang, A. M., Doyle, M. V., and Mark, D. F. (1989) Proc. Natl.

Acad. Sci. USA 86, 9717–9721.4. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D., and 1 Present address: Universitat Wurzburg, Theodor Boveri-Institut

Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184, fur Biowissenschaften (Biozentrum), Abt. Physiologische Chemie II,260–268. AK Prof. Hoppe, Am Hubland, D-97074 Wurzburg, Germany.

2 To whom correspondence should be addressed. Fax: /49-6151-5. de Breuil, R. M., Patel, J. M., and Mendelow, B. V. (1993) PCRMethods Appl. 3, 57–59. 164171. E-mail: di58@pop.th-darmstadt.de.

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NOTES & TIPS 175

MATERIALS AND METHODS

Materials

The Chinese hamster ovary cell line CHO-A11-A279-C7 was kindly provided by Dr. G. Zettlmeissl (Beh-ringwerke, Marburg, Germany). Iodixanol was obtainedas a 60% (w/v) solution in water from Life Technologies(Eggenstein, Germany), SW-41 ultracentrifugationtubes were from Beckman (Munich, Germany), and allother chemicals were from Fluka (Neu-Ulm, Germany).

Methods

Cell culture, preparation of total cell extracts, andsubcellular fractionation. Cells were cultured and to-tal cell extracts prepared as described previously (3).The subcellular fractionation of CHO-A11-A279-C7cells was performed as described previously (4). How-ever, it was necessary to expose cells to hypotonic con-ditions before homogenizing them.

Spectrophotometric assay of iodixanol and determi-nation of densities: Iodixanol standard. An iodixanolstandard with concentrations of 50, 100, 150, 200, 250,and 300 g/liter iodixanol in water was diluted 1:10,000in water and its absorbance at a wavelength of 244 nmwas measured using a Beckman DU-50 spectrophotom-eter with a scanning speed of 500 nm/min. The densityof the standard solutions was plotted against the ab-sorbance of the 1:10,000 diluted standard solutions.

Samples. Subcellular fractions were diluted1:10,000 and their absorbance at 244 nm was mea-sured. The density of the fractions was calculated fromthe absorbance of the diluted sample using the iodixa-nol standard.

RESULTS AND DISCUSSION

The uv spectrum of a iodixanol solution in watershows a maximum at 244 nm and a specific absorptioncoefficient of 320 lrg01cm01 at that wavelength, whichis slightly lower than that of Nycodenz (6).

To evaluate the spectrophotometric determination ofiodixanol at 244 nm an iodixanol standard was pre-pared in the concentration range of 50–300 g/liter, thesame concentration range of iodixanol used to generatedensity gradients. The standard was diluted 10,000-fold in water; the absorbance was measured at 244nm and plotted against the refractive index (2) of theundiluted standard (Fig. 1A). A linear relationship was

FIG. 1. Standard curve and density profile of a iodixanol gradientused for subcellular fractionation of CHO cells. (A and B) An iodixa-nol standard with concentrations of 50, 100, 150, 200, 250, and 300g/liter iodixanol in water was diluted 1:10,000 in water and its ab- linear iodixanol gradient was prepared in the concentration rangesorbance at a wavelength of 244 nm was measured. The absorbance from 100 to 225 g/liter iodixanol using a gradient maker (5), a postnu-of the diluted standard was plotted against the refractive index of clear supernatant from 1.2 1 106 CHO-A11-A279-C7 cells loadedthe undiluted solution (A). The density of the standard solutions onto the gradient and centrifuged at 54,000gav at 47C for 1.5 h. Fifteenwas plotted against the absorbance of the 1:10,000 diluted standard fractions were collected by puncturing the tube at the bottom withsolutions (B). Both lines were calculated by linear regression. (C) A a syringe needle.

AID AB $NAT / 6m21$$222x 12-05-96 21:32:56 abnt

NOTES & TIPS176

2. Ford, T., Graham, J., and Rickwood, D. (1994) Anal. Biochem.obtained (regression coefficient Å 0.9973). Thus, under220, 360–366.the conditions described both are equally suitable for

3. Schroder, M., and Friedl, P. (1996) Biotechnol. Bioeng., in press.determining the density of iodixanol solutions, as is4. Hammond, C., and Helenius, A. (1994) J. Cell Biol. 126, 41–52.demonstrated in Fig. 1B for the absorbance of the di-5. Graham, J., Ford, T., and Rickwood, D. (1994) Anal. Biochem.luted standard. A linear relation (regression coefficient

220, 367–373.Å 0.9967) between the density of iodixanol solutions6. Rickwood, D., Ford, T., and Graham, J. (1982) Anal. Biochem.and the absorbance of their dilutions exists in the con- 123, 23–31.

centration range used to prepare density gradients.Figure 1C shows the density profile of a subcellular

fractionation of CHO-A11-A279-C7 cells determined bythe spectrophotometric assay for iodixanol. This profiledoes not differ from published profiles (4), which were Activity Staining of Protein Inhibitorsdetermined from the refractive index of a mock density of Proteases on Gelatin-Containinggradient. To determine whether this method is specific

Polyacrylamide Gel Electrophoresisfor iodixanol, 1.2 1 106 CHO-A11-A279-C7 cells werelysed in a volume of 300 ml. A 10,000-fold dilution ofthis cell extract exhibited an absorbance of 0.006. As- R. Felicioli,*,† B. Garzelli,* L. Vaccari,* D. Melfi,‡suming the worst case, the accumulation of all cellular and E. Balestreri†material in the fraction with a iodixanol concentration *Dipartimento di Fisiologia e Biochimica, Universita diof 5% (w/v), and not considering the separation of nuclei Pisa, Via S. Zeno, 27. I-56127 Pisa, Italy; †Istituto dibefore performing the subcellular fractionation, this Biofisica, CNR, Via S. Lorenzo, 29. I-56127 Pisa, Italy;would be equal to 3.4% of the absorbance exhibited by and ‡Istituto di Industrie Agrarie, Universita di Pisa,iodixanol. Therefore it was concluded that no interfer- Via S. Michele, 4. I-56124 Pisa, Italyence of cellular material with the spectrophotometricdetermination of iodixanol occurred.

Received August 5, 1996We conclude that the assay presented here can beemployed to determine the density profile of iodixanol

In the past 15 years, since the paper by Heussendensity gradients used for subcellular fractionations.and Dowdle (1) was first published, the electrophoreticThe principle of this assay should be applicable to otherprocedure for the direct visualization of plasminogennonionic density gradient media, such as nycodenz. Aactivators on substrate-containing SDS–PAGE hasspectrophotometric assay for the determination of thebeen successfully used. The major advantage of thisdensity profile of iodixanol gradients provides severalmethod is the action of SDS in semidenaturing the en-advantages: (i) It can be employed on the density gradi-zyme molecules with the result of preventing substrateent on which the sample has been fractionated, re-digestion during the run. After staining and destain-sulting in the use of less nonionic density gradient me-ing, the presence of the proteolytic enzyme is visualizeddium; (ii) no mock fractionation is necessary; (iii) theas a white band in a completely dark blue background.assumption that the mock fractionation and the actualSince large molecular weight protein substrates copoly-fractionation possess identical density profiles is notmerize with the polyacrylamide, and since, after thenecessary; (iv) no refractometer is used, which makeselectrophoresis under semidenaturing conditions,the determination of iodixanol less tedious and moremany proteolytic enzymes (2) are easily renatured onceconvenient for laboratories which lack a refractometer;the denaturant is washed away, the method has been(v) only a small part of each fraction is used for theapplied (3, 4) to many proteolytic enzymes with differ-determination of its density; and (vi) the spectrophoto-ent specificity.metric determination should be less time-consuming

Garcia-Carreno et al. (5) modified this originalthan the determination of refractive indices. Thus, themethod to avoid the use of SDS: after the electropho-spectrophotometric determination of the density pro-retic run under native conditions, the substrate proteinfiles of density gradients made from nonionic densitywas absorbed into the slabs which, after a washinggradient media seems to be a superior alternative tostep, were stained and destained. The authors showedthe determination via the refractive index.the potentiality of this modification in also detecting

Acknowledgment. We thank Dr. G. Zettlmeissl (Behringwerke, soybean trypsin inhibitor (SBTI)1 as a dark blue bandMarburg) for kindly providing the CHO cell line. in a white background by incubating the slabs in a

trypsin bath after the substrate protein had been ab-REFERENCES

1. Rickwood, D. (1983) Iodinated Density Gradient Media: A Practi- 1 Abbreviations used: SBTI, soybean trypsin inhibitor; BBI, Bow-man–Birk inhibitor.cal Approach, IRL Press, Oxford.

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