[민유홍] In vitro caspase 3 assay

In vitro caspase 3 assay

EMBO journal Vol.16 pp.6914-6925, 1997

1.       293T cells are harvested

2.       wash once with PBS

3.       wash once with ice-cold buffer A (20mM HEPES pH7.5, 10mM KCl, 1.5mM MgCl2, 1mM EDTA, 1mM DTT, 0.1mM PMSF)

4.       cell pellet is resuspended in 1 volume of buffer A and incubated on ice for 20min.

5.       cells are broken by passing 20 times through 1ml syringe needle.

6.       centrifuge at 16000 g for 30min 4℃.

7.       transfer supernatant to chilled tube and NaCl is added to a final concentration of 50mM.

8.       pipette 200ul aliquots into chilled tubes. Snap freeze in liquid nitrogen and store at -80℃

9.       Thaw in ice-cold water.

10.    add 10ul of cell extract to tubes containing bovine heart cytochrome c (Sigma # C3131, 1.25 ul of 200uM stock in 50mM phosphate buffer at neutral pH), dATP (Sigma #D6500, 2.5ul of 10mM stock in 50mM Tris pH 7.5) and test amount of a protein diluted in buffer A to 11.25ul.

11.    incubate samples at 30℃ for 60min.

12.    To each well of a 96 well plate, add in the following order, 87.5 ul caspase buffer (50mM HEPES pH 7.5, 100mM NaCl, 1mM EDTA, 0.1% CHAPS, 10% sucrose, 5mM DTT) and 2.5 ul of extracts activated.

13.    Start assay by adding 10ul of 1mM DEVD-AFC in DMSO (100uM final concentration).

14.    Monitor fluorescence (Ex 405nm, Em 535nm, plate definition file COS96fw, gain 60, integration time 60us) at 37℃.