LYSIS BUFFER

(1) Brij Lysis buffer:
For 900 ml, add: Final:
30 ml of 5M NaCl 150mM
25 ml of 1M Tris.HCl (pH7.5) 25mM
10 ml of 500mM EDTA(pH8.0) 5mM

Make fresh for each use: 9 vol buffer + 1 vol of 10% Brij

Add inhibitors before using. For 1ml, add following:
5 μl of 200mM Vanadate Final:1mM (phosphatase inhibitor)
1 μl of 1M AEBSF**(store at 4oC) 1mM (protease inhibitor)
1 μl of 1000x aprotinin (store at 4oC) (protease inhibitor)
1 μl of 1000x leupeptin hemisulfate (protease inhibitor)

*Brij96=polyoxyethylene 10 oleyl ether
**AEBSF (ICN Biochemicals)=4-(2-aminoethyl)-benzen sulfonyl flouride hydrochloride, MW239.7, add 417ml H2O to 100mg to make 1M

Immunoprecipitation

(1) In micro- tube, add 50 μl of protein-A sepharose beads (50%, made in PBS) and antibody

(2) Add lysates. (use about 1 mg protein for coIP).

(3) Rotate, 4° C, 1-1.5 hours.

(4) Wash 4-6 times with lysis buffer (protease inhibitors aren't neccessary at this stage).

(5) Add 50 μl 2x Sample buffer

(6) Heat samples at 95-100°C for 5 min before run on a SDS-PAGE gel.


By: Minghua Zhu