In-Gel Technology provides a more accurate representation of relative protein levels than traditional Western Blotting and it is now optimized for both homemade and precast gels

Pierce In-Gel Chemiluminescent Technology is an alternative to classic Western blotting. With Pierce In-Gel Technology, proteins are detected directly in the gel, eliminating the need to transfer the protein to a membrane. Bypassing the transfer step shortens the complete "blotting" protocol, saving time and money. Use In-Gel Technology and you can proceed from electrophoresis to film in-hand in as little as four hours.

Highlights:

• Uniform representation of antigen(s) not skewed by inefficient transfer
• Compatible with stripping and reprobing protocols
• Transfer and blocking are not necessary
• Sensitive to 1 ng – comparable to ECL Substrate
• Detects nontransferable proteins, providing a more accurate representation of protein levels
• Now optimized for homemade gels; works with more gel types
• Faster than Western Blotting, saving time
• Results in less nonspecific binding than Westerns blots, providing a cleaner image with more specific bands
• Compatible with total protein staining, eliminating the need to run multiple gels

Inconsistent protein transfer

A more important advantage of using Pierce In-Gel Technology is the accurate representation of relative levels of protein in a given sample. Proteins separated by SDS-PAGE need to be transferred to membranes for immunoprocessing. Unfortunately, the transfer process is selective, depending on the nature and size of the proteins (Refs1-3). This selectivity renders any attempts at quantitation of relative proteins levels on a Western blot inaccurate, thus nullifying potentially useful quantitative data.

Experiment demonstrates poor gel-to-membrane transfer of some proteins. Pure GFP/6xHis-tagged protein and E. coli bacterial GFP/6xHis-tagged lysate were separated by SDS-PAGE and then electrophoretically transferred to nitrocellulose membrane. Following the transfer, the protein left in the gel was detected using the Pierce In-Gel System with a 1:500 dilution of anti-Penta His antibody followed by a 1:250 dilution of HRP-labeled goat anti-mouse antibody. Lanes 1-5: E. coli bacterial GFP/6xHis-tagged lysate diluted 1:100, 1:250, 1:1,000, 1:2,000 and 1:4,000, respectively. Lanes 6-13: pure GFP/6xHis-tagged protein at 12.5, 6.25, 3.12, 1.56, 1.0, 0.5, 0.1 and 0.05 ng, respectively. Lane 14: 6xHis-tagged ladder (1:16 dilution).

Total gel staining is possible after immunodetection

Another advantage Pierce In-Gel Technology offers over membrane-based blotting is that In-Gel Technology-detected gels can still be stained for total protein detection after the immunodetection is performed. Because In-Gel Technology detects the proteins directly in the gel, total protein staining with GelCode Blue Stain Reagent (Product # 24592) or any other total protein stain can still be performed. This advantage offers you a great way to demonstrate the specificity of your immunodetection vs. all other proteins in the sample.

Overcoming previous Pierce In-Gel Technology limitations

Pierce scientists previously described a method for detecting antigens directly in a pre-cast polyacrylamide gel using a two-step system (primary antibody/HRP-labeled secondary or biotinylated antibody/Streptavidin-HRP) or a one-step format with a primary antibody conjugated directly to HRP (Refs4,5).

In the past, In-Gel Technology was not consistently compatible with homemade bisacrylamide gels and it was not as sensitive as standard ECL Technology detection. However, we have now optimized a method for detecting antigens in homemade bisacrylamide gels with sensitivity comparable to ECL Technology on-membrane detection. In this new method, homemade gels are poured within siliconized glass plates or within treated disposable plastic cassettes. For gels prepared in siliconized glass plates, a pretreatment with 50% methanol is required. Gels poured within plastic cassettes (Invitrogen) require using 50% isopropanol for the pretreatment. Homemade gels are routinely made with a bisacrylamide polymerization mix with SDS. However, as the presence of SDS in the polymerization mix does not provide any advantages, we recommend eliminating it from the polymerization mix. Gels made without SDS can be eletrophoresed using either native or denaturing conditions.

Pierce In-Gel Technology Detection using Homemade vs. Pre-cast Gels

Figure 3. Direct comparison of GST detection in homemade gels using the Pierce In-Gel Chemiluminescent Detection Kit for GST-tagged Proteins (Product # 33515) and Western Blotting using ECL Western Blotting Detection Reagent. Pure GST proteins and GST lysates were separated by SDS-PAGE in homemade gels. Figure 3A. The gel was cast within siliconized glass plates and pre-treated after electrophoreses with 50% methanol. Figure 3B. The gel was cast within a treated disposable plastic cassette from Invitrogen and pre-treated after electrophoresis with 50% isopropanol. Both gels were processed for immunodetection with Anti-GST-HRP conjugate (0.4 µg/ml). Figure 3C. The gel was transferred to a nitrocellulose membrane after electrophoresis and immunoprocessed with 0.4 µg/ml Anti-GST-HRP conjugate. Lanes 1-3. 10 ng, 25 ng and 50 ng pure GST (Santa Cruz). Lanes 4-7: Id1:GST lysate 1:1, Id2:GST lysate 1:1, Id3:GST lysate 1:10 (BD PharMingen) and Bir2/GST lysate (1:200), respectively.

The binding between the antibody and the antigen within a homemade polyacrylamide gel can be reversed when treated with Restore Western Blot Stripping Buffer (Product # 21059). The stripping buffer removes the primary and secondary antibodies and development reagents from an immunoprocessed gel, allowing reprocess of the gel with different antibodies or different concentrations of the same antibodies. Reprocessing of the gel is useful when samples are limited and optimization of the antibody dilutions is necessary or if analysis of two different antigens using the same gel is desired. It is also possible to probe a homemade gel with a biotinylated antibody and Streptavidin-HRP. Another advantage of the in-gel immunodetection procedure is that the same gel can be washed and stained with GelCode Blue Protein Stain Reagent. An additional gel needs to be prepared if the protein pattern is to be visualized using membrane detection methods.

References:

1. Lin, W. and Kasamatsu, H. (1983).On electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 302-311.
2. DenHollander, N. and Befus, D. (1989). Loss of antigens from immunoblotting membranes. (1989) J. Immunol. Methods 122, 129-135.
3. Reddy V.M. and Kumar B. (2000). Interactions of Mycobacterium avium complex with human respiratory epithelial cells. J. Infec. Dis. 181, 1189-1193
4. Desai, S., Dworecki, B. and Cichon, E. (2001). Direct immunodetection of antigens within the precast polyacrylamide gel. Anal. Biochem. 297, 94-98.
5. Desai, S., Dworecki, B. and Cichon, E. (2002). Direct immunodetection of proteins within polyacrylamide gels. J. Bioluminescence and Chemiluminescence, manuscript accepted for publication.
6. Roberts, K.P., Ensrud, K.M., and Hamilton, D.W. (2002) A comparitive analysis of expression and processing of the rat epididymal fluid and sperm-bound forms of Proteins D and E. Biol. Reprod. 67:525 - 533