CASPASE-3 ACTIVITY ASSAY

SAMPLE PREPARATION:

1. Induce apoptosis in cells by desired method and include a negative control (cells without induction)

2. 2X10⁶ cells are needed per sample

3. Wash cells once in ice cold PBS and centrifuge at 1500 rpm for 3min.

4. Prepare lysis buffer prior to use (1 X DTT (solution 5), 200µl/sample, ice cold)

5. Shortly vortex pellet and then resuspend in 200µl 1 X DTT (solution 5)

6. Incubate for 1 min. on ice

7. Centrifuge for 1 min./+15 to +25^oC at maximum speed in standard table top centrifuge

8. Remove 100 µl supernatant for direct analysis or storage (at-15 to -25^oC for 6 months)

PROCEDURE FOR COATING MTP (modules or plates):

1. Add anti-caspase 3 coating solution (solution 2) 100µl per well

2. Cover the MTP tightly with an adhesive cover foil and incubate either at 37^oC for 1 hour or at 2-8^oC for overnight

3. Remove coating solution by tapping

4. Block unspecific binding by adding blocking buffer (solution3) 200µl and cover the MTP tightly with an adhesive cover foil

5. Incubate at +15 to +25^oC for 30 min.

6. Remove the solution by aspirating the buffer or by tapping

7. Wash three times with solution 4

PROCEDURE FOR ASSAYING PROTEASE ACTIVITY:

1. Add sample (lysate, positive control) 100 µl and cover the MTP tightly with an adhesive cover foil and incubate at 37^oC for 1 hour

2. Remove the lysate by aspirating or tapping

3. Wash three times with solution 4

4. Add 1X substrate solution (6) 100µl, freshly prepared

5. Cover the MTP and incubate at 37^oC for 2 hour

6. Fluorometric measurement:

a. Measure with an excitation filter 370-425 nm and emission filter 490-530 nm (maxima λ_ex=400nm and λ_em=505nm)