To make 1X Cell Lysis Buffer:

Note: This recipe is for 24 samples ONLY- please use accompanying chart for different sample sizes!!

(for 24 samples at 24-well-plate)

(for 24 samples at 12-well-plate)

Home made Cell Lysis Buffer (5X)

125µM Tris-PO₄, 10µM DTT (dithiotretiol), 10µM CDTA (Sigma D0922), 50% Glycerol, 5% Triton x-100)

GME Buffer (1X)

(25mM Glycylglyine (Sigma G2265) , 15mM MgSO₄, 4mM EGTA)

ATP (Sigma A7699)

1g in 4.7ml H₂O

100µl aliquots stored at -20ºC

Protocol

1)      Aspirate media from wells

2)      Wash wells with PBS briefly

3)      Add 400µl/well (12-well/plate) or 200µl/well (24-well/plate) 1X Cell Lysis buffer and lyse for 10-15min while occasionally shaking plate.

4)      Transfer supernatant to a fresh epi tube.

5)      Briefly spin at 13K for 30sec-1min to pellet debris.

6)      Transfer 200µl of supernatant (without contaminating debris) to glass tube for assay.

Note: This recipe is for 24 samples ONLY- please use accompanying chart for different sample sizes!!

Prepare Luciferase Subtrate Solution (Injector 1)

Prepare Mix Solution (Injector 2)