Resources results for "STF-1"
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CIP/Lambda Phosphatase Treatment of Cell Lysates:
1) Wash cells 2X with ice-cold PBS.
2) Add cold 1X cell lysis buffer that does not include phosphatase inhibitors [1-2 E7 cells per ml or 0.4ml per 10 cm plate (80-90% confluent)]. Our cell lysis buffer is prepared as follows:
20 mM Tris-HCl (pH 7.5)
150 mM NaCl
1 mM Na2EDTA
1 mM EGTA
1% Triton
1 ug/ml leupeptin
1mM PMSF
3) Add 20ul Quick CIP (NEB #M0525; https://www.neb.com/products/m0525-quick-cip#Product%20Information) per 400ul of cell extract.
4) Incubate with Quick CIP at 37C for 1 hour.
5) Add 20ul lambda protein phosphatase (CST
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The methods for the LC and MS are shown below:
Parameter | Setting | |
Sample prep | IP sample resuspension volume | 20 uL |
Sample volume per injection | 4 uL | |
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