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 为 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: 2024 年 9 月 12 日
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