Figure 1. 1D optimization of phosphotyrosine Western blotting. The 3 Western blots shown above were obtained by running three standards in triplicate on a single gel in the lane order: sample 1 (EGF stimulated A431 cell prep purchased from Exalpha), sample 2 (EGF stimulated A431 cell prep purchased from Upstate), sample 3 (transformed 3T3 cell prep purchased from Exalpha), MW markers, blank lane. The gel was transblotted to PVDF, the blot stained with Coomassie blue and then divided into thirds through the blank lanes. Each third was Western blotted with a different antibody under identical conditions using ECL technology (GE Healthcare) to visualize results.

The PY20 antibody from Exalpha Biologicals gave an identical pattern to the 4010 clone from Upstate; both were superior to the pattern from the Sigma PT66 antibody. The Exalpha antibody is the best value and our choice for the package offered below. Note that the 1D patterns from the same cultured cell line prepared by different companies were dramatically different (lanes 1 & 2) , suggesting that sample preparation is critical.

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B.  Examples of 2D PTyr Western Blots

The transformed 3T3 cell preparation used above was run on a 2D gel followed by Western blotting. The resultant ECL film in Figure 2 below showed about 10 putative glycosylated proteins characterized by multiple fuzzy isoforms along with 12 proteins showing single, double or triple charge isoforms that would traditionally be expected from phosphorylation. The glycosylated, phosphorylated proteins are probably membrane receptors.

Figure 2. PTyr Western blot from transformed 3T3 cells. A standard format 2D gel was run with just 25 ug of transformed 3T3 cells and Western blotted with the Exalpha PY20 antibody. The ECL detection kit from GE Healthcare was used with x-ray film to visualize the pattern. Only one of the ECL spots could be matched to a faint protein on a Coomassie blue stained duplicate gel (not shown) suggesting that this antibody is quite sensitive.

Figure 3. PTyr 2D Western blot example from client. This ECL film was obtained from mouse embryonic fibroblast after SiRNA knockdown (shown with permission). The 29 thousand MW marker, carbonic anhydrase, is sold as a phosphotyrosine marker by Axxora.

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III. Discussion and References
Genomic methods (microarrays, PCR, SAGE, SNP analysis) have many uses but don't directly reveal what's happening to cellular proteins during disease processes. For this the proteins themselves must be studied. But comparing 2D patterns of cancerous tissue to normal tissue for example, is often unfruitful since numerous proteins from alternate differentiation pathways of the tumor show up as differences. A better proteomics approach is to focus on tyrosine kinase (TK) substrates. As cancer progresses, multiple cumulative mutations occur that probably lead to more and different TKs being sequentially turned on.  Directly analyzing the TK substrate pattern as cancer progresses might identify novel candidates for drug targeting.

We have optimized a method for identifying tyrosine kinase substrates by 2D Western blotting using anti-phosphotyrosine antibody. Preliminary results suggest that the method is specific, reproducible, and sensitive. Anticipated problems are that the proteins of interest will sometimes be in too low abundance to identify by mass spectrometry on a duplicate Coomassie blue stained gel. 

References
1. International Humann Genome Sequencing Consortium, Nature, 431: 931-945, 2004, "Finishing the Euchromatic Sequence of the Human Genome"
2. Z. Su, J. Wang, X. Huang and X. Gu, Genome Res. 16: 182-189, 2006, "Evolution of alternative splicing after gene duplication.
3. Fuchu, He,  Mol. Cell. Proteomics 4.12 1841-48 2005, "Human Liver Proteome Project"  (page 1847)
4. D. Krause and R. Van Etten, NEJM, 353: 172-187, 2005, “Tyrosine Kinases as Targets for Cancer Therapy”
5. R. Weinberg, Book, "Biology of Science", ISBN-10: 0815340788, 2006
 

INTRODUCTORY OFFER 40% OFF: PHOSPHOTYROSINE WESTERN BLOTTING PACKAGE INCLUDING PY20 ANTIBODY.
Price: Standard format: BP-7SF: $300/sample includes standard format 2D gel electrophoresis, transblotting to PVDF, blot Coomassie staining, subsequent immunostaining with PY20 antibody, and electronic photos of stained blot and ECL film. BP-7SF-Dup: replicate W blot package $150 each. BP-7LF large format package: $375, BP-7SF-Dup: large format replicate W blot $190 each.  

Mass spectrometry to identify proteins and phosphorylation sites found by Western blotting is extra and requires a duplicate Coomassie blue stained gel. Ask for a quote by calling 800-462-3417 or emailing 2d@kendricklabs.com

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