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immunoprecipitation with anti-pS and anti-pT

Any questions have been raised for the use of anti-phosphoserine antibodies.
First of all, the anti-pS and anti-pT antibodies are much less affinity than
anti-pY (usually in 100 nM to 1 uM range). Therefore the interaction is
extemely concentraion depependent. The conventional IP protocol is to add
the anti-pS to the cell lysate and incubate overnight then precipitated with
Protein A. This approach has greatly dilute the anti-pS concentration. Our
approach is: pack a 10 uL protein A beads in a pepette tip (glass wool as
filter); pass the anti-pS (10 ug) through the pipette tip several times and
blocked with 1% BSA. Then pass the 500 ug of crude cell lysate through the
the anti-pS-protein A pipette tip three times. Wash the beads with PBSt
several time or remove the glass wool to release the beads to a Eppendoff
vial for washing, using preimmune serum as control. Using such emthod, we
have immunopreciptiate several kinase such as p44-MAPK, RSK, and S6K, while
no kinase were immunoprecipitated with control.
In this case, anti-pS concentration is over 2 mg/mL (10 uL beads has only 4
uL void space), while the phosphoprein concentration remains the same.
Anti-pS recognizes the phosphopeptide well better than the phosphoprotein,
suggesting the sterichindrance.