Western- SuperStar Immunodetection Systemtools.thermofisher.com/content/sfs/manuals/cms_049536.pdf · The Western-SuperStar Immunodetection System is composed of the Western-SuperStar

Western-SuperStar™ Immunodetection SystemChemiluminescent Immunoblot Detection System

with SuperStar™ Substrate

P/N 4388983, 4388984

Protocol

Western-SuperStar™ Immunodetection System

Table of Contents

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

A. Product DescriptionB. System Components and Storage ConditionsC. Equipment and Reagents Not Provided with the SystemD. Related Products Available from Applied Biosystems

II. Immunodetection Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

A. Buffer PreparationB. Primary and Secondary Antibody IncubationsC. Chemiluminescent Detection Procedure

III. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

A. Lower Than Expected Protein Detection SensitivityB. High Nonspecific Background

IV. Associated Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

A. Immunodetection of a Control ProteinB. Protocol Modifications for Nylon MembraneC. Stripping Antibodies and Reprobing

V. Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

A. ReferencesB. Safety Information

Part Number 4388980 Revision A Revision Date: January 29, 2008

For research use only. Not for use in diagnostic procedures.

Information in this document is subject to change without notice. Applied Biosystems assumes no responsibil-ity for any errors that may appear in this document.

Applied Biosystems disclaims all warranties with respect to this document, expressed or implied, including butnot limited to those of merchantability or fitness for a particular purpose. In no event shall Applied Biosystemsbe liable, whether in contract, tort, warranty, or under any statute or on any other basis for special, incidental,indirect, punitive, multiple or consequential damages in connection with or arising from this document,including but not limited to the use thereof.

Literature Citation: When describing a procedure for publication using this product, please refer to it as theWestern-SuperStar™ Immunodetection System.

Warranty and Liability: Applied Biosystems is committed to delivering superior product quality and perfor-mance, supported by industry-leading global service and technical support teams. Warranty information forthe accompanying consumable product is available at www.ambion.com/info/warranty in “Limited Warrantyfor Consumables,” which is subject to the exclusions, conditions, exceptions, and limitations set forth underthe caption “EXCLUSIONS, CONDITIONS, EXCEPTIONS, AND LIMITATIONS” in the full warrantystatement. Please contact Applied Biosystems if you have any questions about our warranties or would likeinformation about post-warranty support.

Trademarks: Applied Biosystems, AB (Design), Ambion, CSPD, CDP-Star, and Tropix are registeredtrademarks, and Western-SuperStar, Blot-Loc, mirVana, PARIS, ELISA-Light, Sapphire-II, Emerald-II,Nitro-Block-II, I-Block, Western-Light, Western-Star and NeoFX are trademarks of Applera Corporation or itssubsidiaries in the US and/or certain other countries.

All other trademarks are the sole property of their respective owners.

© 2008 Applied Biosystems, Inc. All Rights Reserved.

I.A. Product Description

Introduction

1

I. Introduction

A. Product Description

The Tropix® Western-SuperStar™ Immunodetection System is a nextgeneration chemiluminescent detection kit for immunodetection offemtogram amounts of protein immobilized on membranes. Like itspredecessors, the Western-Light™ and Western-Star™ systems, theWestern-SuperStar Immunodetection System provides consistent, reli-able results. With its rapid, flexible detection capability and increasedsignal intensity, the Western-SuperStar system presents distinct advan-tages over other systems.

Proprietary

chemiluminescent

SuperStar™ Substrate

The Western-SuperStar Immunodetection System employs the propri-etary next generation, 1,2-dioxetane chemiluminescent substrate,SuperStar™ Substrate, for rapid, sensitive protein detection. The chemi-luminescent signal is detectable immediately and persists for 24 hourswith low background and consistent sensitivity at any timepoint. Thelong-lived signal enables results to be collected either immediately afteradding the SuperStar substrate, or up to an entire day later.

The Western-SuperStar Immunodetection System uses standard west-ern blot methodology for immunodetection of proteins using anti-gen-specific primary antibodies and secondary antibodies conjugated toalkaline phosphatase (AP). Alkaline phosphatase is a preferred enzymelabel due to its stability, insensitivity to azide, and high sensitivity ofdetection when coupled with 1,2-dioxetane chemiluminescent sub-strates. Upon dephosphorylation by alkaline phosphatase, the SuperStarSubstrate decomposes, producing a prolonged emission of light that canbe imaged on X-ray film or with a CCD camera imaging system, to pro-vide a permanent record of results. Figure 1 on page 3 shows an over-view of the Western-SuperStar Immunodetection System procedure.

High quality reagents and

your choice of secondary

antibodies

The Western-SuperStar Immunodetection System is composed of theWestern-SuperStar Reagent Set and your choice of either Goatanti-Mouse IgG+IgM or Goat anti-Rabbit IgG AP-conjugated second-ary antibodies. The reagent set includes ready-to-use Western-SuperStarBlocker and SuperStar Substrate, as well as wash and assay buffer con-centrates. The system enables immunodetection of both proteins andphosphorylated epitopes using the same protocol and reagents. Theblocking agent provided does not contain phosphoproteins, and allbuffers are Tris-based. Finally, the system includes convenient tools,such as Tweezers and Blot-Loc™ blot processing bags that reduce back-ground caused by contaminated membrane processing trays and exces-sive handling, and Development Folders to conveniently image blots.


I.A. Product Description2

Membrane compatibility The Western-SuperStar Immunodetection System is compatible withboth PVDF and nitrocellulose membranes. (Nitro-Block-II™ chemilu-minescence enhancer is provided for use with nitrocellulose membrane.)It is also possible to use most of the kit components for detection of pro-teins immobilized on neutral nylon membrane, however, alternativewash and blocking solutions are needed (see section IV.B on page 12).

Applications The Western-SuperStar Immunodetection System is well-suited for allapplications involving immunodetection of proteins from both culturedcells and tissues (Berger 2003) immobilized on a membrane, including“dot-blotting” applications (Shoda 2001) and protein array detection(Sack 2005). With its improved signal intensity and rapid detection, theWestern-SuperStar Immunodetection System is particularly well-suitedfor applications in which available sample volumes are limiting, such asRNAi-mediated protein knockdown validation and stem cell analysisworkflows (Erickson-Miller 2000, Wegmuller 2007).

Recent reports in the scientific literature cite the use of Applied Biosys-tems Tropix® brand chemiluminescent Western-Star and Western-LightSystems in RNAi-mediated protein knockdown workflows. Extractsfrom cells treated with siRNA or other mediators of RNAi, includingmiRNA, are subjected to standard SDS-PAGE techniques followed bywestern immunodetection to evaluate knockdown of the target protein(Grunweller 2003, Forler 2004, Behm-Ansmant 2006, Rehwinkel2006, and Eulalio 2007). This technology has been used for confirma-tion of protein capture via immunoprecipitation (Hakki 2006), as wellas protein identification following cell surface biotinylation and affinitypurification (Chang 1995, Kojro 2001). These immunodetection sys-tems have been used for detection of phosphorylated protein epitopes(Smith 2001, Hakki 2006) and for detection of epitope-tagged proteins(Kojro 2001, Hakki, 2006). In addition, these immunodetection sys-tems can be used in conjunction with biotinylated secondary antibodies,using a streptavidin-alkaline phosphatase conjugate for the final detec-tion step. The Western-SuperStar methodology also enables highly sen-sitive biotinylated protein detection (Gillespie 1991, Araga 2000).

I.A. Product Description

Introduction

3

Figure 1. Western-SuperStar™ Immunodetection System Procedure

1° 1°

1° 1°

2° 2°

AP AP

2°

APlight

SuperStar™Substrate

1. Rinse blot briefly in 1X Wash Buffer

2. Incubate blot in Western-SuperStar Blocker for 30–60 min

3. Incubate blot with primary antibody diluted in Western-SuperStar Blocker for 30–60 min

4. Wash blot 2 x 5 min in 1X Wash Buffer

6. Wash blot 2 x 5 min in 1X Wash Buffer

5. Incubate with 2nd Antibody-AP diluted 1:5000 in Western-SuperStar Blocker for 30–60 min

1. Drain blot and place it on a development sheet

7. Wash blot 2 x 2 min in 1X Assay Buffer

2. Pipette SuperStar Substrate onto blot and incubate 5 min

3. Drain excess SuperStar Substrate and image blot

Primary and Secondary Antibody Incubations

Chemiluminescent Detection Procedure


I.B. System Components and Storage Conditions4

B. System Components and Storage Conditions

This kit contains reagents to process 20 minigel protein blots(≤100 cm2 each).

Western-SuperStar

Immunodetection Systems

The Western-SuperStar Immunodetection System consists of the West-ern-SuperStar Reagent Set and an anti-mouse or an anti-rabbit second-ary antibody conjugated to alkaline phosphatase (2nd Antibody-AP).

Western-SuperStar Reagent

Set

AP-conjugated secondary

antibodies

Secondary antibodies are supplied in 50% glycerol. Just before use, therequired amount must be diluted in Western-SuperStar Blocker.

P/N Product

4388983 Western-SuperStar Reagent Set w/ Goat Anti-Rabbit IgG (H&L)

4388984 Western-SuperStar Reagent Set w/ Goat Anti-Mouse IgG+IgM (H&L)

Amount Component Storage

650 mL Western-SuperStar™ Blocker• Provided ready-to-use

2–8°C

220 mL Western-SuperStar™ 10X Wash Buffer• 10X TBS, 1% Tween-20, pH 7.4• Dilute 1:10 with deionized water before use.

2–8°C

150 mL 10X Assay Buffer• 200 mM Tris (pH 9.8), 10 mM MgCl2• Dilute 1:10 with deionized water before use.• Also available separately, P/N T2187

2–8°C

75 mL SuperStar™ Substrate• Provided ready-to-use

2–8°C

5 mL Nitro-Block-II™ Enhancer• For use with nitrocellulose membranes only• 20X chemiluminescence enhancer, dilute 1:20

with SuperStar Substrate before use.

2–8°C

20 ea. Blot-Loc™ Bags Ambient*

* Alternatively, these components can be left in the box for storage at 2–8°C.

30 ea. Development Folders• Also available separately, P/N T2258

Ambient*

1 ea. Tweezers Ambient*

Amount Component Storage

100 μL 2nd Antibody-AP, Goat Anti-Mouse IgG + IgM• Also available separately, P/N T2192

2–8°C

100 μL 2nd Antibody-AP, Goat Anti-Rabbit IgG• Also available separately, P/N T2191

2–8°C

I.C. Equipment and Reagents Not Provided with the System

Introduction

5

C. Equipment and Reagents Not Provided with the System

• Protein blot prepared on PVDF (preferred) or nitrocellulose mem-brane; we recommend Millipore Immobilon-P membrane. Blotsprepared on neutral nylon membrane are also compatible with theWestern-SuperStar Immunodetection System, but require alternatewash and blocking buffers as described in section IV.B on page 12.

• Rabbit or mouse primary antibody to the protein of interest. UseWestern-SuperStar Immunodetection System P/N 4388983 withprimary antibodies derived from rabbit and P/N 4388984 for pri-mary antibodies derived from mouse.

• Rocking or rotating platform mixer• High quality, ultrapure, deionized water • X-ray film and film processing system, or CCD-camera based imag-

ing system

D. Related Products Available from Applied Biosystems

Primary Antibodies for siRNA

ResearchCat #AM4300, AM4302, AM4309

Primary antibodies corresponding to select Ambion Silencer Control and Val-idated siRNAs, such as Anti-GAPDH, Anti-β-actin, and Anti-Cyclophilin AAntibodies are ideal for use in Western blots to detect siRNA-induced knock-down at the protein level. The GAPDH mouse monoclonal antibody targetsGAPDH in human, rabbit, mouse, fish, frog, and chicken.

PARIS™ KitCat #AM1921

The PARIS Kit (Protein and RNA Isolation System) uses a fast, simple proce-dure to recover both RNA and native protein from the same experimentalsample. In addition, samples can be separated into nuclear and cytoplasmicfractions prior to RNA and/or protein isolation. The protein and RNAobtained using the PARIS Kit are suitable for most downstream applications,reducing time, cost, and variability for experiments that require analysis ofboth protein and RNA.

mirVana™ PARIS™ KitCat #AM1556

The mirVana PARIS Kit employs a unique and versatile procedure for quan-titative recovery of native protein and all RNA species, including small RNAssuch as microRNA (miRNA), small interfering RNA (siRNA), small nuclearRNA (snRNA), and small nucleolar RNA (snoRNA), from the same sample.The kit also includes a procedure to enrich the population of small RNAs(<200 nt) which can enhance sensitivity in downstream applications.

ELISA-Light™ Immunoassay

SystemP/N T1022, T1023, T1024, T1025,

T1026

The ELISA-Light™ Immunoassay System employs an alkaline phosphataseenzyme label for ultra-sensitive detection of antigens or antibodies in multipleimmunoassay formats. Kits contain reagents optimized for chemiluminescentELISAs with ready-to-use chemiluminescent substrate formulationscontaining CSPD® or CDP-Star® substrate with Sapphire-II™ or Emerald-II™luminescence enhancers.


II.A. Buffer Preparation6

II. Immunodetection Protocol

A. Buffer Preparation

WARNING

CHEMICAL HAZARD. Western-SuperStar 10X Wash Buffer may

cause eye, skin, and respiratory tract irritation. It may be harmful if swal-

lowed, inhaled, or absorbed through the skin. Avoid breathing vapor. Use

only with adequate ventilation. Avoid contact with eyes and skin. Do not

taste or swallow. Read the MSDS, and follow the handling instructions.

Wear appropriate protective eyewear, clothing, and gloves.

CHEMICAL HAZARD. 10X Assay Buffer causes eye, skin, and respira-

tory tract irritation. It may be harmful if swallowed, inhaled or absorbed

through the skin. Avoid breathing vapor. Use only with adequate ventilation.

Avoid contact with eyes and skin. Do not taste or swallow. Read the MSDS,

and follow the handling instructions. Wear appropriate protective eyewear,

clothing, and gloves.

Dilute the supplied 10X

Wash and Assay Buffers to

1X

Prepare only enough buffer for blots to be processed that day. This min-imizes the chance for buffers to become contaminated.

Western-SuperStar 10X Wash Buffer

Dilute Western-SuperStar 10X Wash Buffer to 1X final concentrationwith deionized water and mix well.For one minigel blot (≤100 cm2), you will need 100 mL 1X WashBuffer: add 10 mL Western-SuperStar 10X Wash Buffer to 90 mLdeionized water.

10X Assay Buffer

Dilute 10X Assay Buffer to 1X final concentration with deionized waterand mix well.For one minigel blot, you will need 40 mL 1X Assay Buffer: add 4 mLof 10X Assay Buffer to 36 mL deionized water.

B. Primary and Secondary Antibody Incubations

IMPORTANT

Membranes must be handled carefully and should never be touched with

bare hands.

Perform all steps at room temperature with gentle agitation on a rocking or

rotating platform mixer.

NOTE

The volumes in this procedure are appropriate for a single minigel blot

(≤100 cm2). They will require adjustment for blots of other sizes.

II.B. Primary and Secondary Antibody Incubations

Immunodetection Protocol

7

1. Rinse blot briefly in

1X Wash Buffer

Following electrotransfer of proteins to the membrane, rinse blot brieflyin ~20 mL 1X Wash Buffer.

2. Incubate blot in

Western-SuperStar

Blocker for 30–60 min

a. Open a Blot-Loc Bag fully (it may help to gently shake the bag), andadd 10 mL Western-SuperStar Blocker to the bag.

b. Using the supplied Tweezers, carefully insert the blot into the bag (seeFigure 2). Then remove the air from the bag and seal it by firmlypressing the zip lock channels together.You may want to make a small mark on the bag to identify the ori-entation of the membrane.

c. Incubate for 30–60 min with gentle agitation on a rocking or rotatingplatform.

NOTE

In all of the following steps, slowly pour off the solution in the bag and add

the next solution directly to the bag.

3. Incubate blot with

primary antibody diluted

in Western-SuperStar


a. Dilute antigen-specific primary antibody according to the supplier’srecommendations in ~5–10 mL Western-SuperStar Blocker.

b. Pour the solution from the bag and discard, add diluted primaryantibody. Reseal the bag, removing bubbles as much as possible.

c. Incubate for 30–60 min with gentle agitation.

4. Wash blot 2 x 5 min in

1X Wash Buffer

a. Pour the solution from the bag and discard. Add ~20 mL 1X WashBuffer, and reseal.

b. Wash blot for 5 min with gentle agitation.

c. Repeat steps 4.a and 4.b to wash a second time.

Figure 2. Placing Blots into Blot-Loc™ Bags

To prevent the blot from sticking to the bag as you insert it into theBlot-Loc Bag, grasp two corners of the blot with the tweezers without creas-ing the blot and slide the membrane into the bag without touching thesides and allow it fall open. Use the tweezers only on the edges of the blot.


II.C. Chemiluminescent Detection Procedure8

5. Incubate with 2nd

Antibody-AP diluted

1:5000 in

Western-SuperStar


a. Dilute 2 μL of secondary antibody-AP (either 2nd Antibody-AP, GoatAnti-Mouse IgG + IgM or 2nd Antibody-AP, Goat Anti-Rabbit IgG) in10 mL in Western-SuperStar Blocker (final dilution is 1:5000).

b. Pour the solution from the bag and discard. Add diluted secondaryantibody and reseal.

c. Incubate for 30–60 min with gentle agitation.


1X Wash Buffer

Wash the blot 2 x 5 min with ~20 mL 1X Wash Buffer as in step 4.


1X Assay Buffer

a. Pour the solution from the bag and discard. Add ~20 mL 1X AssayBuffer and reseal.

b. Wash the blot for 2 min with gentle agitation.

c. Repeat steps 7.a and 7.b to wash a second time.

C. Chemiluminescent Detection Procedure

1. Drain blot and place it on

a development sheet

a. Cut the fold-line from a Development Folder to generate twodevelopment sheets as shown in Figure 3.

b. Place one of the sheets on a smooth, flat surface with the interior sidefacing up. This will provide a clean, convenient surface for SuperStarSubstrate incubation. Reserve the other sheet, with the anti-staticprotector, for future use.

c. Carefully remove the blot from the Blot-Loc Bag using Tweezers andtouch a corner of the blot to a clean paper towel to drain away excesssolution.

d. Place the blot on the development sheet with protein-side facing up.Proceed immediately to the next step so that the blot does not dry.

Figure 3. Cutting a Development Folder into Two Sheets

II.C. Chemiluminescent Detection Procedure

Immunodetection Protocol

9

2. Pipette SuperStar

Substrate onto blot and

incubate 5 min

CAUTION

CHEMICAL HAZARD. Nitro-Block-II Enhancer may cause eye, skin,

and respiratory tract irritation. It may be harmful if swallowed, inhaled or

absorbed through the skin. Avoid contact with eyes and skin. Do not taste or

swallow. Read the MSDS, and follow the handling instructions. Wear appro-

priate protective eyewear, clothing, and gloves.

PVDF membrane: Pipette a thin layer of SuperStar Substrate (~3 mL)onto the blot and incubate for 5 min. (Do not use Nitro-Block-IIEnhancer with PVDF membrane.)

Nitrocellulose membrane: Add 150 μL Nitro-Block-II Enhancer to3 mL of SuperStar Substrate, and mix well by gentle vortexing. Pipettethe mixture over the blot to form a thin layer and incubate for 5 min.

3. Drain excess SuperStar

Substrate and image blot

a. Drain excess SuperStar Substrate solution by touching a corner of theblot to a paper towel for a few seconds.

b. Remove the anti-static sheet from a fresh uncut Development Folderand place the blot inside.

c. Image the blot with standard X-ray film or a CCD-camera basedimaging system. Initial exposures of 1 sec to 1 min are recommended(see Table 1). Blots can be imaged immediately, up to 24 hr later after SuperStarSubstrate incubation.

Table 1. X-ray Film or CCD-Camera Imaging Times

Membrane Imaging Time

PVDF 1 sec – 1 min

Nitrocellulose*

* Developed using SuperStar Substrate with Nitro-Block-II™ Enhancer.

1 sec – 5 min


III.A. Lower Than Expected Protein Detection Sensitivity10

III. Troubleshooting

This protocol has been optimized using the reagents supplied in theWestern-SuperStar Immunodetection System. Since the SuperStar Sub-trate provides extremely sensitive detection, it is recommended thatonly ultrapure water and other reagents free of alkaline phosphatase andbacterial contamination be used. Below are suggestions for commonwestern blotting technical difficulties.

A. Lower Than Expected Protein Detection Sensitivity

Inadequate exposure time Increase film or CCD camera imaging time.

Poor quality primary

antibody

The sensitivity and specificity of western blotting can only be as good asthe primary antibody used. If sensitivity is low using a primary antibodythat has not been tested in your lab previously, consider trying a primaryantibody from a different source.

Problems with antibody

incubation

• Increase the concentration of primary antibody. Be aware that thismay increase nonspecific background. Antibody titering is alwaysrecommended for optimal results.

• Confirm that the secondary antibody you are using is appropriate foryour primary antibody. For example if the primary antibody is frommouse, be sure to use the anti-mouse secondary antibody (labeled2nd Antibody-AP, Goat Anti-Mouse IgG + IgM).

• Increase incubation time with your primary antibody or the suppliedAP-conjugated secondary antibody.

• Membrane blocking (section II.B, step 2) and/or incubation in pri-mary antibody solution (section II.B, step 3) may be extended toovernight at 4°C to reduce background and increase specific signal.

Problems with protein

transfer to the membrane

• Ensure that protein transfer to the membrane was adequate by stain-ing the original gel with an appropriate protein stain to visualize anyresidual proteins. Modify the transfer method if a large number ofbands are seen on gels after protein transfer to the membrane.

• Optimize PAGE and/or electrotransfer conditions.

B. High Nonspecific Background

Blot is overexposed Decrease the exposure time. To optimize blot exposure time, start witha 1 sec exposure, and then perform a series of exposures with increasingtimes, for example, 3 sec, 10 sec, 30 sec, ~1 min, and finally 5 min if theblot is on nitrocellulose membrane (1 min is typically the maximumusable exposure time for PVDF membrane).

III.B. High Nonspecific Background

Troubleshooting

11

Poor quality primary

antibody

The sensitivity and specificity of western blotting can only be as good asthe primary antibody used in the procedure. If you are using a primaryantibody that has not been tested in your lab previously and blots showhigh nonspecific background, consider using a primary antibody from adifferent source.

Bacterial contamination of

reagents

Splotchy, spotted, or pebbled images may result from bacterial contam-ination of one or more reagents. To prevent bacterial contamination,use sterile pipets when removing reagents from stock bottles. Also, storereagents and buffers as indicated on their labels and prepare wash bufferand assay buffer fresh daily using the highest quality water possible.

Incubation times require

optimization

• Increase wash times (section II.B, steps 4, 6, and 7) to as long as15 min.

• Membrane blocking (section II.B, step 2) or incubation in primaryantibody solution (section II.B, step 3) may be performed overnightat 4°C to reduce background and increase specific signal.

Antibody dilutions require

optimization

With chemiluminescent immunodetection, it is typically possible anddesirable to use more dilute primary and secondary antibody concentra-tions than with other detection chemistries.• Optimize the dilution of the primary antibody.• Increase the dilution of the secondary antibody to 1:10,000–50,000.

Blots were damaged by

handling

Blots should always be handled gently, on the edges only, with theTweezers provided. The Blot-Loc bags and reagents should always behandled using clean gloves.


IV.A. Immunodetection of a Control Protein12

IV. Associated Protocols

A. Immunodetection of a Control Protein

Strategies for detection of

two proteins on the same

blot

The level of a control protein, such as a housekeeping protein, can bemonitored to control for even sample loading across a gel and betweengels. There are two main ways to do this:1. If the housekeeping and experimental proteins migrate to distinct

positions on the gel during electrophoresis, then typically, both pro-teins can be immunodetected at the same time. To do this, followthe instructions in section II. Immunodetection Protocol starting onpage 6, but add primary antibodies against both the housekeepingprotein and the experimental protein simultaneously in section II.B,step 3 on page 7. Note that if primary antibodies for the two proteinsare derived from the same species, a single secondary antibody-APconjugate in step 5 on page 8, can detect both primary antibodies. Ifdifferent secondary antibody-AP conjugates are needed, they canalso be incubated with blots simultaneously.

2. To detect proteins that are not well separated by PAGE, immunode-tect the experimental protein first, following the default protocol insection II. Immunodetection Protocol starting on page 6. Then stripthe blot following the instructions in section IV.C. Stripping Anti-bodies and Reprobing on page 13, and reprobe the blot for the controlprotein following the routine procedure, starting at section II.B, step2 on page 7.

Using Ambion® anti-GAPDH Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a commonlyused control protein in western blotting. Ambion anti-GAPDH, mousemonoclonal 6C5, Cat #AM4300 is ideal for detection of GAPDH as ahousekeeping protein loading control. Use it at a dilution of 1:4000 insection II.B, step 3 on page 7 and incubate with 2nd Antibody-AP,Goat Anti-Mouse IgG + IgM in section II.B, step 5 on page 8.

B. Protocol Modifications for Nylon Membrane

The Western-SuperStar Immunodetection System can be used withneutral nylon membrane, but the wash and blocking solutions providedwith this kit are not compatible with nylon membranes. Instead, use thewash and blocking solutions described below. Because nylon mem-branes typically require casein-based blocking reagent for optimalresults, they are not recommended for phosphoprotein detection.

Wash solution for nylon

membranes

In place of the Western-SuperStar Wash Buffer supplied with the kit,use a solution of 1X PBS, 0.1%Tween-20 throughout the protocol.

IV.C. Stripping Antibodies and Reprobing

Associated Protocols

13

I-Block™ blocking solution

for nylon membranes

In place of the Western-SuperStar Blocker supplied with the kit, useI-Block blocking solution, following the instructions below.

Prepare at least 150 mL I-Block blocking solution for 100 cm2 of mem-brane as follows.1. Add 15 mL 10X PBS to 135 mL deionized water.2. Microwave for 40 sec, then slowly add I-Block™ reagent while stir-

ring on a hot plate. Continue to heat and stir the solution until allparticles are dissolved. Do Not Boil.

3. Add Tween®-20 detergent after solution has cooled. The solutionwill remain slightly opaque.

4. Cool to room temperature before use.

C. Stripping Antibodies and Reprobing

NOTE

The success of the stripping and reprobing procedure may vary depending

on membrane, primary antibody, antigen, and use of Nitro-Block enhancer.

Optimization of the stripping conditions and/or using an alternative stripping

protocol can improve results (Kaufmann 1992).

Stripping antibodies 1. Incubate blot 2 x 30 min in 10 mL glycine stripping buffer (seeTable 3) at room temperature with gentle agitation.

2. Wash 3 x 5 min with 1X Wash Buffer (at least 20 mL per wash).

Table 2. I-Block™ Blocking Solution

Final Concentration Amount for 150 mL

1X PBS 15 mL 10X PBS

0.2% I-Block™ Reagent*

* Applied Biosystems P/N T2015

0.3 g

0.1% Tween®-20 150 μL

Table 3. Glycine Stripping Buffer

Final ConcentrationAmount for

100 mL

0.2 M Glycine 1.5 g

0.1% SDS 0.1 g

1% Tween®-20 Detergent 1 mL

Add the reagents shown above to 80 mL deionized water.Adjust pH to 2.2 with HCl.Adjust volume to 100 mL with deionized water.


IV.C. Stripping Antibodies and Reprobing14

NOTE

For the MSDSs of chemicals not supplied by Applied Biosystems, contact the

chemical manufacturer.

Reprobing a stripped

membrane

To immunodetect a stripped blot, simply follow the routine procedure,starting at section II.B, step 2 on page 7.

V.A. References

Appendix

15

V. Appendix

A. References

Araga S, et al. (2000) A peptide vaccine that prevents experimental autoimmune myasthenia gravis by specifi-cally blocking T cell help. FASEB J. 14: 185–196.

Behm-Ansmant I, et al. (2006). mRNA degradation by miRNAs and GW182 requires both CCR4:NOT dead-enylase and DCP1:DCP2 decapping complexes. Genes & Development 20: 1885–1898.

Berger A, et al. (2003) Severe depletion of mitochondrial DNA in spinal muscular atrophy. Acta Neuropathol.105: 245–251.

Chang A et al. (1995) α3β1 and α6β1 Integrins mediate laminin/merosin binding and function as costimula-tory molecules for human thymocyte proliferation. J. Immunol. 154: 500–510.

Erickson-Miller C, et al. (2000) Signaling induced by erythropoietin and stem cell factor in UT-7/Epo cells:transient versus sustained proliferation. Stem Cells 18: 366–373.

Eulalio, A, et al. (2007) P-Body Formation Is a Consequence, Not the Cause, of RNA-Mediated Gene Silenc-ing. Mol. Cell. Biol. 27 (11): 3970–3981.

Forler D, et al. (2004) RanBP2/Nup358 provides a major binding site for NXF1-p15 dimers at the nuclearpore complex and functions in nuclear mRNA export. Mol Cell Biol 24(3): 1155–1167.

Gillespie PG and Hudspeth AJ (1991) Chemiluminescence detection of proteins from single cells. Proc. Natl.Acad. Sci. USA 88: 2563–2567.

Grunweller A, et al. (2003) Cellular uptake and localization of a Cy3-labeled siRNA specific for the serine/thre-onine kinase Pim-1. Oligonucleotides 13: 345–352.

Hakki M, et al. (2006) Binding and nuclear relocalization of PKR by human cytomegalovirus TRS1. J Virol(JVI) 80(23): 11817–11826.

Kaufmann SH and Shaper JH (1992) Erasable western blots. In Methods in Molecular Biology: Immunochem-ical Protocols (Manson M, ed.), Vol. 10, pp. 235–246. Humana Press, Inc.

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V.B. Safety Information16

B. Safety Information

The MSDS for any chemical supplied by Applied Biosystems orAmbion is available to you free 24 hours a day.

IMPORTANT

For the MSDSs of chemicals not distributed by Applied Biosystems or

Ambion, contact the chemical manufacturer.

To obtain Material Safety

Data Sheets

1. Go to www.appliedbiosystems.com, click Support, then clickMSDS Search.

2. In the Keyword Search field, enter the chemical name, productname, MSDS part number, or other information that appears in theMSDS of interest and then click Search.

3. Find the MSDS of interest, click the link or right-click the MSDStitle, then select any of the following: • Open – To view the MSDS• Print Target – To print the MSDS• Save Target As – To download a PDF version of the MSDS

Chemical safety guidelines To minimize the hazards of chemicals:• Read and understand the Material Safety Data Sheets (MSDS) pro-

vided by the chemical manufacturer before you store, handle, orwork with any chemicals or hazardous materials.

• Minimize contact with chemicals. Wear appropriate personal protec-tive equipment when handling chemicals (for example, safety glasses,gloves, or protective clothing). For additional safety guidelines, con-sult the MSDS.

• Minimize the inhalation of chemicals. Do not leave chemical con-tainers open. Use only with adequate ventilation (for example, fumehood). For additional safety guidelines, consult the MSDS.

• Check regularly for chemical leaks or spills. If a leak or spill occurs,follow the manufacturer’s cleanup procedures as recommended onthe MSDS.

• Comply with all local, state/provincial, or national laws and regula-tions related to chemical storage, handling, and disposal.

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www.appliedbiosystems.com Part Number 4388980 Rev. A

01/2008

Documents

Western- SuperStar Immunodetection Systemtools.thermofisher.com/content/sfs/manuals/cms_049536.pdf · The Western-SuperStar Immunodetection System is composed of the Western-SuperStar