A: Protein lysis buffer components:
what do you think of my lysis buffer preparation?
1) 1M Hepes (pH 7.5) stock:
11.915g hepes
Add 45ml miliQ
Adjust pH, top up with miliQ TO 50ml
2) 5M sodium chloride:
14.61g sodium chloride
Top up till 50ml miliQ
*might take more than a day to dissolve, use Mei hua’s stock solution if urgent
3) 0.5M EGTA (pH 8):
1.902 g EGTA
Add 8ml miliQ
Adjust pH, top up with miliQ to 10ml
4) 1M B-glycerol phosphate:
10.8g b-glycerol phosphate
Top up till 50ml miliQ
5) 0.1M Sodium orthovanadate
Take 0.9196 g sodium orthovanadate
Top up till 50ml miliQ
6) 0.5 M Sodium fluoride
Take 1.0497g Sodium fluoride
Top up till 50ml miliQ
7) 20% triton X
Measure 10ml triton, top up till 50ml miliQàaluminum wrap falcon tube
*leave on roller RT overnight
8) 1M MgCl
4.761g
Top up till 50ml miliQ
B: On day of lysis
prepare 10ml fresh lysis solution from reagents made in A):
250ul of 1M hepes
600ul of 5M NaCl
10ul of 1M Mgcl2
20ul of 0.5M EGTA
200ul of 1M b-gycerol phosphate
100ul of 0.1M sodium vanadate
200ul of 0.5M sodium fluoride
574 ul of glycerol
250ul of 20% triton
7.8 ml of PBS
Add in DTT & protease inhibitor:
C: On day of lysis
Take 958ul from above solution
1ul of 1M DTT
41.6ul of protease inhibitor
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