Thermo Scientific Pierce In-Gel Chemiluminescent Detection Kits comprise an innovative variation on traditional Western blot analysis by making it possible to probe and detect proteins directly in the gel.
In-Gel Chemiluminescent Technology provides an alternative to traditional Western blotting. Proteins are detected directly in the gel, eliminating the need to transfer the protein to a membrane. This method circumvents the transfer and blocking steps entirely, enabling immunoblotting techniques to be applied to proteins that cannot be transferred efficiently from a gel to a membrane.
Highlights:
- No transfer required – eliminates the quantitative skewing of results as a result of uneven or inefficient transfer or protein binding
- No blocking step – method circumvents traditional blocking steps and other variables associated with membrane blotting
- Sensitive – detects bands containing at least 1ng protein, comparable to standard ECL formulations
- Compatible – method is effective with several types of precast gels, as well as homemade protein gels
- Versatile – detected gel can be stripped and reprobed, or it can be stained by traditional methods
Product Details:
An 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, 1:1000, 1:2000 and 1:4000, 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 0.05ng, respectively. Lane 14: 6xHis-tagged ladder (1:16 dilution). |
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| Direct comparison of GST detection in homemade gels using the Therm Scientific Pierce In-Gel Chemiluminescent Detection Kit 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 electrophoresis 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 (0.4µg/mL). Figure 3C. The gel was transferred to a nitrocellulose membrane after electrophoresis and immunoprocessed with 0.4µg/mL Anti-GST-HRP conjugate. Lanes 1-3. 10ng, 25ng and 50ng 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. |
References:
- Lin, W. and Kasamatsu, H. (1983).On electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 302-311.
- DenHollander, N. and Befus, D. (1989). Loss of antigens from immunoblotting membranes. (1989) J. Immunol. Methods 122, 129-135.
- Reddy V.M. and Kumar B. (2000). Interactions of Mycobacterium avium complex with human respiratory epithelial cells. J. Infec. Dis. 181, 1189-1193
- Desai, S., Dworecki, B. and Cichon, E. (2001). Direct immunodetection of antigens within the precast polyacrylamide gel. Anal. Biochem. 297, 94-98.
- Desai, S., Dworecki, B. and Cichon, E. (2002). Direct immunodetection of proteins within polyacrylamide gels. J. Bioluminescence and Chemiluminescence, manuscript accepted for publication.
- 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
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