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I. Solutions:
a. Chelsky Buffer (200ml)
0.01M Tris-HCl
0.01M NaCl
0.003M MgCl2
0.03M Sucrose (mw. 342.3 g/mole)
pH to 7.0 and bring up to 200ml.
b. NP-40 Buffer
100ml of Chelsky Buffer
500ul of NP-40 detergent
c. Cacl2 Buffer
100ml of Chelsky Buffer
1.0ml 100x stock CaCl2 sol'n (10mM)
d. Buffer C
20mM Tris-HCl pH 7.9
20% glycerol
0.1M KCl
0.2mM EDTA
pH to 7.9
II. Procedure:
a. Cell collection:
Plate Helas on 150mm plates on day 1. (1 x107 cells yields about 1 mg of total cellular protein) Approximately 32-150mm plates.
Trypsinize every plate on day 2-3 (am).
Bring each plate up in 5ml of media, and count the total number of cells\ml. Record the volume of cell suspension.
b. Cell treatment:
Note: Everything (buffers, cells, centrifuge) must be kept on ice (4oC) throughout this entire procedure, to reduce protein degradation.
Note: Save the supernatant from each wash to keep track of protein if lost.
Wash the Hela cells 2x with 25 ml of cold PBS per tube in clinical centrifuge setting #3 in cold room(4oC).
Resuspend cell pellets in 2.0 ml of NP-40 Buffer, spin in HB-40 rotor at 3000rpm (1500xg) for 10mins,remove supernatant and resuspend as above.
Repeat NP-40 wash step above.
Remove supernatant and resuspend pellet into 2.0ml of CaCl2 Buffer.
Spin in HB-40 rotor at 3000 rpm (1500xg) for 10mins, remove supernanat and resuspend as above. Repeat CaCl2 wash step.
Resuspend pellet from second CaCl2 wash step into 2.0 ml of Buffer C, depending on how much protein was collected (use 1ml/number of cells)
Spin balanced tubes in SS34 rotor at 14,500 rpm(25,000xg) for 30mins.
Collect supernatant (nucleoplasm), resuspend pellet (nuclear envelope) in Buffer C and homogenize to get into solution.
Aliquot into cryovials and freeze at -70oC.