Phosphotyrosine Western Blotting
OUTLINE
I. Why focus on
phosphotyrosine Western blotting?
II.
1D optimization
III. 2D examples
IV. Discussion, References
V. Introductory Offer - package price
I. Why
focus on Phosphotyrosine Western blotting?
Although the human genome contains 20,000-25,000 protein coding genes plus
many variants from alternative splicing (1, 2),
the number of expressed proteins/cell type is much less, around 5,000
in liver for example (3). Our silver-stain resolves 800-1200 polypeptide spots
on large format gels, but misses low abundance proteins. Western blotting,
up to
100 times more sensitive than silver staining, focuses on subsets of low
abundance proteins.
Phosphotyrosine Western blotting focuses on a very
important subset of proteins that control cell growth and division. Nonproliferating cells have
very low levels of tyrosyl phosphorylated proteins (4) while various
tyrosine kinases (TK) are turned on during cell division. For example,
Robert Weinberg in "Biology of Cancer" (5) discusses numerous TK oncogenes
that have been mutated to be
activated during cancer, and also the success of Gleevec, a TK inhibitor. Over
13,600 other articles containing the keyword "phosphotyrosine" have
been published from 2000 through July 2007 so this area is hot. Given this importance, we
decided to put time and resources into optimization of phosphotyrosine
Western blotting as a service.
We started with optimization on 1D slab gels. Three commercially available mouse monoclonal antibodies against phosphotyrosine
were chosen: 1. Sigma-Aldrich Clone PT-66, 2. Upstate Clone
4010, and 3. ExalphaBio Clone PY20. Figure 1 shows a comparison of the three
antibodies under identical conditions.

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.
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.
Return to top
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
Return to top
Go to Catalog