[molecular biology work] Preparation_of_cytoplasmic_extracts_and_gel

Preparation of cytoplasmic extracts and gel-filtration from 293 cells

 

 

1.     spin down the cells at 600Xg for 5min and wash with 1X ice cold PBS

( 150 mm 5dishes)

2.     resuspend the cells with 2ml hypotonic buffer

3.     stand the cells on ice for 30 min.

4.     pass the cells with the 3ml syringe

( kink the needle and careful not to foam)

5.     spin down the cell lysates at 26,000Xg for 20min.

6.     take the sup and add equal volume of 150mM buffer

(careful not to aggregate the proteins)

7.     Quantitate the protein and concentrate with 9mg/ml

8.     spin down the lysates and load the sample(0.5ml) to superdex 200 HR 10/30 equilibrated with 80mM KCl buffer.

(0.2ml/min, 0.25ml/frac)

9.     harvest the fractions and load each fraction with the volume of 50ul to SDS-PAGE

**** unless described, all steps are performed at 4 degree****

 

Hypotonic Buffer

10mM HEPES, 10mM KCl, 1.5mM MgCl2, 0.5mM EGTA, pH 7.6, 1mM PMSF, 5ug/ml aprotinin, 12mM b-glycerophosphate, 1mM sodium-orthovanadate, 10mM NaF, 1mM DTT.

 

Instead of 10mM KCl, Add 80mM or 150mM KCl