Transcript anti-Phosphoprotein ab

Talk Outline
 Excerpt from talk given Nov 06 at
the American Electrophoresis
Society meeting in San Francisco
 P-Serine & P-Threonine Western
blotting with ECL Advance from
GE Healthcare
1
2D gels contain TMI (too much
information). How to simplify?
Focus on subsets.
 Subcellular fractions
Mitochondria, nuclei, microsomes etc.
But often the subcellular fractions are
complex too.
 Phosphorylated proteins using
Western blotting
Antibodies give an enormous increase in
sensitivity, 10-100X silver staining.
2
The human genome has
518 protein kinases
Phosphorylated proteins mediate:
 cell division (cancer)
 cellular differentiation
 hormonal signal transduction
3
Western blot protocol
1. Perform 1D or 2DE on a sample prepared in the
presence of protease and phosphatase inhibitors;
transfer the proteins to PVDF.
2. Wet the blot in methanol, rinse, block in 5% BSA.
3. Incubate with anti-Phosphoprotein ab overnight.
4. Wash 3X.
5. Incubate the PVDF in secondary ab (goat anti-mouse
HRP conjugated) for 2 hr with shaking.
6. Wash 3X.
7. Use GE Healthcare’s enhanced chemiluminescence
reagent (ECL or ECL-Advance) to detect signal.
4
P-Tyrosine Westerns work well,
but we currently have more calls
for P-Serine & P-Threonine
Western blotting than P-Tyr.
• Approximately 30% of cellular
proteins are phosphorylated,
most at serine and threonine
residues (Philip Cohen, TIBS 25, 596, 2000),
5
P-threonine and
P-serine antibodies are
weak and expensive!
Company
Antigen
Dilution
Cost/2Dblot
Abcam
phospho-serine
1:500
$330
Abcam
phospho-threonine
1:200
$330
Typical mouse monoclonal antibodies for Western blotting
Many companies sell these abs.
6
Results have been poor. Typical results with
ECL for 1:200 dilution antibodies are shown
below. (Coomassie stained PVDF on left; ECL
film on right)
7
ECL Advance from
GE Healthcare
GE web site says ECL Advance sensitivity is
pg, much greater than ECL, so primary ab
concentrations may be reduced.
8
We chose Qiagen antibodies:
• PhosphoSerine ab Q5 (IgG & IgM) for
detection of phosphorylated serine residues, irrespective of
surrounding amino acids
• PhosphoThreonine ab Q7 (IgG)
for detection of phosphorylated threonine residues,
irrespective of surrounding amino acids
Both are $340 for 100 ul; suggested dilution
is 1:200, enough for 1 2D gel.
9
Tries 1-9 with 1D
optimization using rat liver
homogenate are not shown.
Try 10, 2DE, next slide…..
10
Qiagen Anti-PSer versus Anti-PThr for 2D
gels from same rat liver homogenate
These 2D patterns are excellent. Similarities between the patterns
would be expected since it is known that the P-Ser & P-Thr kinases
are not very selective.
11
Anti-PSer + anti-PThr combined:
The abs were combined before the dilutions. 1:4000 clearly gives a
darker yet clean pattern. (Secondary ab was not changed.)
This is what we’re currently using for clients.
12
Summary
 ECL-Advance gives a dramatic
increase in sensitivity for P-Serine
and P-Threonine Western blotting.
It’s a major breakthrough.
 We’re not sure of the specificity of
the Qiagen antibodies. They don’t
pick up casein for example. We
can’t be positive that all proteins
detected are phosphorylated.
 Differences between control and
test samples should be valid even if
some nonspecific binding occurs.
13