Phosphotyrosine Western Blotting
1D optimization
Discussion, References
Prices for package
including 2D gel, antibody, Western blotting and electronic photos.
Why optimize PTyr Western blotting? Tyrosine
phosphorylation is an important post-translational modification involved in
many aspects of cell growth and differentiation. For example, nonproliferating cells have
very low levels of tyrosyl phosphorylated proteins while dividing
cells have activated tyrosine kinases (TK) that generate relatively high
levels of phosphorylated species (1). TK oncogenes have been shown to drive cell division during
various kinds of cancer (2). Elisa's and kinase arrays exist to measure
tyrosine kinases but there are few ways to measure unknown TK substrates.
Thus we
decided to optimize phosphotyrosine
Western blotting and provide it as a service.
Figure 1. Anti-phosphotyrosine Western blot from transformed 3T3 cells
(purchased). 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. The ECL film shows 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.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. The large presumptive bovine serum albumin
(BSA) protein spot from the culture media is negatively staining.
Figure 2. Anti-phosphotyrosine 2D Western blot from an
erythroid progenitors sample. The PVDF blot was immunostained with the Exalpha
PY20 antibody under standardized conditions. The ECL detection kit from
Pierce was used with x-ray
film to
visualize the pattern. A longer film exposure was performed to reveal more
detail. Shown with permission of Dr. Amittha Wickrema, University of
Chicago.
Figure 3. Anti-phosphotyrosine 2D Western blot using the
PY20 antibody obtained from mouse embryonic fibroblast after SiRNA knockdown The ECL
detection kit from Pierce was used with x-ray
film to
visualize the pattern (shown with permission). The 29 thousand MW marker, carbonic anhydrase, is sold as a phosphotyrosine marker by Axxora.
Figure 4. Anti-phosphotyrosine 2D Western blot using the
PY20 antibody against rat retinal pigment, eye epothelium. The ECL detection
kit from Pierce was used with x-ray film to visualize the pattern.
1D Optimization showed the Exalpha antibody was the best value.
Three commercially available mouse monoclonal antibodies against phosphotyrosine
were tested: 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 Western blotting conditions for three
commercially available samples.
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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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. Comparing
complex 2D patterns of diseased to normal tissue is one way to study
protein changes but the results will be confusing if cellular
differentiation is involved. Numerous proteins from alternate
differentiation pathways, for example, tumor versus adjacent normal liver
sample, will show
up as differences. A better proteomics approach is to focus on tyrosine
kinase (TK) substrates to elucidate novel pathways that differ between
samples.
We have optimized a method for identifying tyrosine kinase substrates
by 2D Western blotting using an 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. D. Krause and R. Van Etten, NEJM, 353: 172-187, 2005, “Tyrosine Kinases as Targets for Cancer Therapy”
2. Robert A. Weinberg, "The Biology of Cancer", Garland Science, New
York, NY, 2007.
PHOSPHOTYROSINE WESTERN BLOTTING PACKAGE INCLUDING PY20 ANTIBODY.
Price: Standard format: BP-7SF: $325/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 $175 each.
BP-7LF large format package: $400,
BP-7SF-Dup: large format replicate WB package $220 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
Note that PTyr WB patterns
may change after samples sit frozen for several weeks. Duplicate blots at
the onset are better than retesting later.
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