West lab Anti-pMeCP2 (Ser421) antibody notes
July 2011
This purified peptide antibody was raised in rabbit against the peptide
CEKMPRGGpSLESD encompassing Ser421 of mouse MeCP2. The antibody recognizes
mouse, rat, and human MeCP2, we have not tested other species.
The antibody has very high sensitivity for phosphorylated MeCP2. On brain sections and
in cultured cells we see very robust +/- staining for pMeCP2. Unlike the
punctate/heterochromatin pattern of total MeCP2, pMeCP2 fills the whole nucleus,
though there is often one brighter spot. The aliquot provided should be diluted 1:1000 for
immunofluorescence on brain sections or fixed cells in culture. Information is provided
below regarding western analysis. On brain sections the antibody easily detects the
Ser421 phosphorylated form of MeCP2. Specific immunoreactivity is absent when the
antibody is tested on the MeCP2-308 hypomorphs (which lack the C-terminal domain of
MeCP2 containing the 421 peptide) or MeCP2-Ser421Ala knockin mice.
For brain sections our staining protocol is outlined in Deng et al (2010) Nat Neurosci
13:1128. Briefly, mice are perfused with 4% PFA, then brains are removed and post-fixed
overnight in PFA and sucrose. If storage is required, we store the brains in PBS at 4
degrees. We process quickly once they are cut. We cut 40µm thick sections on a freezing
microtome, then process as floating sections. For pMeCP2 we permeabilize first for 1
hour in 1% Tx-100 in PBS then block for 1 hour in 16% goat serum with 0.3% Tx-100 in
PBS. The antibody is diluted in the block solution and incubated overnight at 4 degrees.
The high percent triton X-100 helps to get the antibody into the nuclei but will diminish
staining for some cytoplasmic markers. Longer primary antibody incubations can help
increase the pMeCP2 signal if it is not possible to do the 1% Tx-100 extraction.
Our antibody works somewhat less well on western and controls are required to ensure
the signal is specific for phospho MeCP2. We believe this is due to the relative affinity of
the antibody for the phosphorylated versus the nonphosphorylated peptide. Slot blot
analysis shows that the antibody has >100 fold preference for the phosphorylated peptide
on western versus the nonphosphorylated form. However the preference is not absolute.
This is not a problem on brain sections or cell culture because each cell seems to either
have pMeCP2 or to lack pMeCP2. However in lysates of brain tissue from samples in
which only a small percentage of cells have pMeCP2, but all cells have MeCP2, the
cross-reactivity of the antibody with the non-phospho peptide can be a concern. In
addition this antibody has some cross reactivity with non-nuclear proteins that can
obscure the signal on western. This again is not an issue on brain sections because we
quantify only nuclear protein (overlaps with Hoechst). There are three ways we cope with
these issues.
First, we always use nuclear extracts, not total cell lysates, to detect pMeCP2 from brain
samples on western. This improves the signal to noise substantially. Our nuclear extract
protocol follows. You can also run nuclear extracts from MeCP2 null mice to identify
specific versus non-specific bands.
Second it is essential to titrate the concentration of the antibody used on western with
positive and negative controls. For these control samples we make nuclear extracts from
cultured neurons under two conditions – 1) treated overnight with TTX and 2) stimulated
for 60 minutes with 55mM KCl in an isotonic solution (see below). The TTX treated cells
should have little to no pMeCP2 and the KCl treated cells should have high levels of
pMeCP2. We test these samples with dilutions of the antibody (1:1000, 1:3000,
1:10,000, 1:30,000 and 1:100,000) to choose the dilution that gives the best signal to
noise ratio.
Finally, in addition to using the pMeCP2 antibody to detect the phosphorylated form of
MeCP2, we also run SDS-PAGE gels under conditions that allow us to resolve the
phosphorylated form of MeCP2 as a more slowly migrating band with the total MeCP2
antibody. To achieve this, nuclear extracts are run on 8% gels very slowly (100mV) until
the 75kD band is nearly at the bottom of the gel. The TTX/KCl nuclear extracts should
clearly show only one band (TTX) and two bands (KCl). The top band is the Ser421
phosphorylated form of MeCP2 and should co-migrate with the pMeCP2+ band. I have
not yet seen a brain sample that shows as robust of an upward shift as you see in cultured
cells with KCl. This may be due to the presence/absence of other phosphorylation sites
influencing the shift or it may be because we have not yet found an in vivo condition that
induces phosphorylation in as many cells as we can get in cultured cells with KCl.
Nonetheless, seeing the upward smear provides a nice independent confirmation of the
signal seen with the pMeCP2 antibody.
Nuclear Extract protocol:
1. Take up cells in 1-5mL lysis buffer OR dounce tissues in 10-25mL lysis buffer.
2. Spin low speed, 1500xg (3500-4000 RPM on GSA, 1000 RPM in a clincal centrifuge),
5’, 4°C. Remove supe by hand (keep if you want the cytosolic fraction).
3. Resuspend gently in low salt buffer about 1-10mL. Shake tube by hand to resuspend
pellet. Spin 1500xg (1000RPM clinical centrifuge, 3800 RPM eppendorf
minicentrifuge), 5’, 4°C. Remove supe by hand and get all supe off.
4. Resuspend nuclear pellet in an equal volume of high salt buffer (about 0.1-1.0 mL).
Vortex hard. Let sit on ice for 30-60’ vortexing occasionally.
5. Spin in tabletop ultra, 0.5mL per tube (14 tubes max) at 50,000RPM, 30-60’, 4°C in
TLA 120.1.
6. Take off the clear supe leaving behind the thick pellet. Store at –80.
7. Protein assay with Pierce DTT compatible kit. I get about 1ug/uL from tissue, to
100ng/uL for cell culture.
8. Lysis buffer (for 50mL)is:
0.5mL Hepes 1M pH7.9
0.17 mL 3M KCl
0.1mL 1M MgCl2
2.5 mL 10% NP40
150uL 1M DTT
46.6 mL ddH2)
10mM
10mM
2mM
0.5%
3mM
Also to all solutions just before using add protease inhibitors (i.e.: PMSF,
aprotinin, etc) and phosphatase inhibitors (i.e.: sodium fluoride 1mM, sodium
orthovanadate, beta-glycerolphosphate 1mM)
9. Low salt solution (for 10mL) is:
200uL Hepes 1M pH7.9
3.1mL 80% glycerol
20uL 1M MgCl2
0.33 mL 3M KCl
20uL 0.5M EDTA
50uL DTT 1M
6.45 mL ddH2O
20mM
25%
2mM
0.1M
1mM
5mM
10. High salt solution (for 10mL) is:
200 uL Hepes 1M pH7.9
3.1mL 80% glycerol
20uL 1M MgCl2
3.3 mL 3M KCl
20uL 0.5M EDTA
50uL 1M DTT
3.35 mL ddH2O
20mM
25%
2mM
1M
1mM
5mM
Depolarization solution:
Membrane depolarization solution:
For 500mL, to 450mL ddH2O add
170mM KCl
6.33g KCl
10mM Hepes pH 7.4
1.19g Hepes
1mM MgCl2
101mg MgCl2
2mM CaCl2
147mg CaCl2
pH to 7.4 with NaOH and make to 500mL
Add 0.45mL Depolarization solution to 1mL of medium to make an iso-osmotic
55mM extracellular KCl solution.