Extract Preparation Protocol

Cytoplasmic Extract Protocol Sheet

Date:_____________________________
Cell type:__________________________
Media:____________________________
Cells/ml:___________________________
Culture volume (ml):_________________
Treatment before extraction:
(eg TPA -4hr)

Take 10ml sample for RNA analysis.

Spin down cells at 300g in Sorvall

Resuspend pellet in ice cold 1 x PBS (15-50ml)

Transfer to conical tube and spin 300g in benchtop centrifuge, 5 min

Resuspend pellet in ice cold 1 x PBS (15-50ml)

Spin 300g in benchtop centrifuge, 5 min

Note packed cell volume:

Packed Cell Volume:_______ µl

Resuspend in 5PCV buffer A + DTT, PMSF

Buffer A [KCl]:___________ mM

Buffer A swelling (5 x PCV)________ ml

Swell on ice 10 min

Spin 300g in benchtop centrifuge, 5 min

Carefully remove supernatant. Add 2PCV Buffer A

Buffer A lysis (2 x PCV)__________ ml

Lyse by douncing 10 times on ice with loose pestle.

Spin 2000rpm in benchtop centrifuge, 5 min

Take the supernatant to a fresh tube. Estimate extract volume.

Extract volume:_________ µl

Add buffer B (0.11 vol for 10mM buffer A, 0.09 vol for 50mM buffer A)

Buffer B volume:_________ µl

Spin 100000g for 1 hour.

Remove supernatant to fresh tube. Estimate extract volume.

Extract volume:__________ µl

Add 0.25 volumes 80% glycerol.

80% glycerol volume:_________ µl

Final extract volume:__________ µl

Final extract concentration:_________ mg/ml

Storage location:___________

 

Tested for activity

Date:

Method:

Tested for activation

Date:

Method: