Can you share a protocol for treating cell lysates with CIP/Lambda Phosphatase?

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 #89726; https://www.cellsignal.com/product/productDetail.jsp?productId=89726) and 40ul 10 mM MnCl2 per 400ul of cell extract.

6) Incubate with lambda phosphatase at 30C for 1 hour.

7) Add SDS sample buffer to 1X and boil for 3-5 minutes.

8) Load gel or store extracts at -20C.

Last updated: September 12, 2024