Detection Principle and Experimental Procedure for Caspase Activity Assay Kit (by Spectrophotometry)

Detection principle：

Caspase (Cysteine-requiring Aspartate Protease) is a protease family that plays an important role in the process of apoptosis. Caspase exists in the form of prozyme in the normal state and has no activity. However, during the apoptosis stage, activated Caspase which cleavages the corresponding substrate of endochylema or cytoplasmic nuclear and eventually leads to apoptosis.

Caspase Activity Assay kit is used to conjugate Caspase sequence-specific peptides acetyl-peptide(Ac-peptide) to yellow group p-nitroaniline (pNA). When the substrate is cut by Caspase, the yellow group pNA is dissociated. pNA has an absorption peak at 405nm. Measure the OD value at 405 nm and then Caspase activity can be calculated accordingly.

Reagents not included:

PBS, Protein Quantitative Kit (Bradford, optional)

Experimental Procedure:

1. Reagent preparation：

1)Take out the Lysis Buffer ,dissolve fully, mix it and put on ice for use.

2)Lysis working solution preparation: Take 50 μL Lysis Buffer ,add 0.5 μL DTT , to the Lysis Buffer, mix it and put on ice for use.

2. Sample preparation

1)Suspension cells

a)Induce apoptosis of suspension cells with reagents of interest, centrifuge at 2,000 rpm for 5 min, discard the supernatant. Add appropriate PBS to resuspend gently and count the cells.

b)Centrifuge at 2,000 rpm for 5 min, discard the supernatant. Add 50 μL cold Lysis Buffer working solution to each 2 million cells to resuspend the cells. Incubate in ice bath for 30 min and oscillate 3~4 times during incubation.

2)Adherent cells

a)Adherent cells should be detached with trypsin and then collected sedimentary cells. Collect the cells and centrifuge at 2,000 rpm for 5 min, discard the supernatant. Add appropriate PBS to resuspend gently and count the cells.

b)Centrifuge at 2,000 rpm for 5 min, discard the supernatant. Add 50 μL cold Lysis Buffer working solution to each 2 million cells to resuspend the cells. Incubate in ice bath for 30 min and oscillate 3~4 times during incubation..

3)Tissue

a)Take 50 mg tissue, cut to small pieces, then add 200 μL cold Lysis Buffer working solution and homogenize the sample on ice.

      b)Transfer the tissue homogenate to a 1.5 mL centrifuge tube, then incubate in ice bath for 5 min.

c)Centrifuge at 12,000 rpm for 10~15 min at 4°C.

d)Take the supernatant to a new tube, put it on ice for test.

e)Carry out the assay immediately or store the samples at-70°C. Meanwhile, you could also determine the concentration of protein with Bradford method [E-BC-K168, please contact the local distributors].

3. Caspase 3 activity detection

1)Take Ac-peptide-pNA and 2 ×Reaction Buffer, dissolve fully and put on ice for use.

      2)2 ×Reaction working solution preparation: Add 0.5 μL DTT to each 50 μL 2 ×Reaction Buffer.

3)Take 45 μL cell lysate or supernatant of tissue homogenate (contain 100~200 μg of protein), if the volume is less than 45 μL, add lysis buffer to 45 μL.

Operation table

Tips:

1. Add 2 ×Reaction working solution into the tube firstly, then add Sample or Lysis working solution. Mix and avoid bubble formation.

2. Add Ac-peptide-pNA into the tube, Mix it and avoid bubble formation.

4)Incubate at 37℃ for 2~4 h. Measure the OD value (A₄₀₅) at 405 nm with spectrophotometer (100 μL cuvette) or microplate Reader when the color changes obviously. The reaction time can be extended or stay overnight if the color doesn't change significantly.

5)Calculate OD_Sample /OD_Blank to determine the activity of Caspase 3.

Definition: One unit Caspase 3 activity is amount of enzyme that will cleave 1.0 nM of the colorimetric substrate Ac-peptide-pNA per hour at 37℃ under saturated substrate concentrations.