Caspases,Paracaspases, and Metacaspases Methods and Protocols

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should be used as controls. If assessing caspase-2 activity in protein

extracts, controls should also include untreated cells and apoptotic

cell extracts.

1. To assess the activity of recombinant caspase-2 protein, add
   100 nM purifi ed caspase-2 to Caspase-2 Assay Buffer supple-
   mented with 100 μM VDVAD-AMC (or AFC-conjugated
   substrate) in a total volume 100 μl.


2. To monitor caspase-2-like activity in apoptotic protein extracts,
   add cell extract containing 20–50 μg total protein to Caspase-2
   Assay Buffer supplemented with 100 μM VDVAD-AMC (or
   AFC-conjugated substrate) in a total volume 100 μl.


3. As a control, recombinant caspase-2 protein should be prein-
   cubated with inhibitor peptide for 30 min at room tempera-
   ture, prior to the addition of peptide substrate.


4. Monitor cleavage by measurement of AMC/AFC fl uorescence
   every 10–15 min over 1–2 h on a spectrophotometer at 37 °C.
   AMC fl uorescence is measured at excitation wavelength
   360 nm and emission wavelength 460 nm (AFC fl uorescence:
   excitation 400 nm; emission 505 nm).


5. Plot the data as relative fl uorescence units versus time (min) for
   each sample, and calculate the slope of the line from the linear
   part of the curve ( see Note 7 ).


6. Caspase activity is expressed as picomole substrate hydrolyzed/
   min/μg protein. To do this, generate an AMC/AFC fl uores-
   cence calibration curve by measuring fl uorescence of each of
   the AMC/AFC calibration standards (serial dilutions with
   concentration range 0–50 μM) made in assay buffer (fi nal vol-
   ume 100 μl). Plot relative fl uorescence units (RFU) versus
   AMC/AFC concentration (μM) and calculate the slope of the
   linear region for the calibration standard.


7. Calculate

Caspase activity

pmol slope of sample RFU

slope o

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/

min min

1

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assay volume l amount of prot

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m eeinused()mg

To validate caspase-2 substrates and/or determine caspase-2 acti-

vation, in vitro qualitative assays can be carried out with recombi-

nant caspase-2 incubated with either in vitro-translated protein

substrates or cell extracts.

1. The cDNA encoding the specifi c caspase-2 substrate is cloned
   in plasmid vectors that carry either SP6, T3, or T7 promoters
   (e.g., pBluescript, pGEM-T, or pcDNA3 vectors). If it is not
   possible to clone the entire protein coding region, truncated

3.3 Assessing
Caspase-2 Activity by
Cleavage of Labeled
Substrates

3.3.1 Cleavage
of^35 S-Met Labeled
Caspase-2 Substrates
In Vitro

Loretta Dorstyn and Sharad Kumar

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