assay protocol

Caspase-3 activity assay

Caspases play important roles in inducing apoptosis in which caspase-3 is an executive molecule. Caspase-3 has many functions in various pathways via which apoptosis signals transduce. Caspase-3 normally exists in cytoplasm in form of pro-enzyme. In initiation stage of apoptosis, caspase-3 is activated. Activated caspase-3 is composed of two subunits: 17kD one and 12kD one, and it lyses substrate in cytoplasm and nucleus. In the ending stage of apoptosis, the activity of caspase-3 significantly declines.

1. Western-blot assay: analysis towards activation of pro-caspase-3

(Sample western blot result shown below the protocol)

Protocol:

  • Harvest cultured cells with lysis buffer.
  • Quantify total protein concentration by BCA assay. (BCA assay protocol)
  • Isolate target protein by SDS-PAGE. (SDS-PAGE protocol)
  • Transfer proteins to nitrocellulose or PVDF membranes.
  • Incubate in blocking buffer (PBST or TBST with 1% bovine serum albumin or 1% non fat milk), for 20 minutes to 1 hr at RT or 37°C.
  • Incubate with 1:1000~1:2000 of caspase-3 monoclonalantibody (MAb) or polyclonal antibody (PAb) at RT for 1~2h or at 4°C for overnight.
  • Wash the membrane with PBST or TBST for 3*10min
  • Incubate with 1:5000~1:10000 of anti-mouse IgG secondary antibody conjugated with HRP or AP at RT for 30min~1h
  • Wash the membrane with PBST or TBST for 4*15min
  • Develop with ECL or NBT/BCIP
caspase-3

2. Fluorescence spectrometer assay

Principle:

Activated caspase-3 can specificly cleave substrate of D1E2V3D4-X by hydrolyzing peptide bond of D4-X. Based on this reaction, Ac-DEVD-AMC, a short peptide coupled with fluorescent dye was designed. In covalent coupling, AMC can't be activated to eject fluorescence. After the short peptide is hydrolyzed in which AMC is released, free AMC can be activated to eject fluorescence. According to the fluorescence intensity released by AMC, caspase-3 activity can be measured.

fluorescence AMC

Protocol:

  • Harvest cultured cells with lysis buffer
  • Add in Ac-DEVD-AMC (caspase-3 tetrapeptide substrate) and incubate at 37°C for 1h
  • Place in influorescence spectrometer to analyze influorescent intensity. (excitation wavelength: 380nm, emission wavelength: 430-460nm)

3. Flow cytometry assay

Protocol:

  • Harvest cultured cells with lysis buffer
  • Add in Ac-DEVD-AMC and incubate at 37°C for 1h
  • Place in a UV flow cytometer to calculate caspase-3-positive cells and average fluorescence intensity.