II. Caspase Assays (Colorimetric and Fluorometric) The colorimetric caspase substrates can be used to measure the induction of caspase activity in apoptotic cells, or to screen for activators and inhibitors of caspases.
This is a general protocol designed for use with a microplate reader. The amounts of cell extract, substrate, inhibitor, and p-nitroaniline (pNA) used are for an assay volume of 140 ml. The researcher may scale up this procedure to use in a spectrophotometer. Optimization is recommended. Solutions, Reagents, and Equipment
Caspase Substrate
Caspase Inhibitor
Cell Lysis Buffer: 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 1 mM DTT, 100 mM EDTA, pH 7.4
Assay Buffer: 50 mM HEPES, 100 mM NaCl, 0.1% CHAPS, 10 mM DTT, 100 mM EDTA , 10% glycerol, pH 7.4
PBS (phosphate buffered saline): Dissolve 8 g of NaCl, 200 mg of KCl, 1.44 g of Na2HPO4 in 800 ml of distilled water; adjust the pH to 7.4 with HCl; add distilled water to 1 L
96-well plate and 96-well plate reader Recommended Additional Materials
Purified Caspase
p-Nitroaniline (pNA)
Preparation of Cell Extracts
Induce apoptosis in cells by using an appropriate apoptosis inducing agent (see previous protocol). Controls may include untreated cells, cells treated with an inactive analog of the apoptosis inducer (if available), or a “time-zero” sample from the apoptosis induction time course. A sufficient number of cells must be used to assay caspase activity in duplicate, and must include an inhibitor control to determine protein concentration. In general, the number of cells described below will give an adequate protein concentration for the 96-well plate assay; however, differences in cell size, volume, and protein concentration may necessitate increased plating densities. When 2 x 107 cells/ml are used for lysis, the protein concentration will be about 1-3 mg/ml and a 10 ml assay sample will contain 10-30 mg of protein. Depending on the cell line, a minimum of 106 cells in 50 ml lysis buffer are required.
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Count cells and harvest by centrifugation. Wash cells one time with PBS. If the cells have been treated with a potential caspase inhibitor, it may be necessary to wash more thoroughly to prevent any adverse effects.
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Resuspend cells to the desired concentration using ice-cold lysis buffer. Incubate 5 minutes on ice.
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Centrifuge at 10,000 x g, 10 minutes at 4°C.
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Save the supernatant (cytosolic extract) and hold on ice until use. Extracts can be flash-frozen in an acetone/ethanol bath and stored at -70°C for later use.
Assay Procedure
Preparation of stock solutions of caspase substrate and inhibitor.
1.
Prepare a 100 mM stock solution of substrate in DMSO. Prepare a 1:50 dilution (2 mM) of the stock solution in assay buffer. Aliquot and freeze the remaining stock solution at -20°C.
2.
Prepare a 100 mM stock solution of inhibitor in DMSO. Prepare a 1:1000 dilution (100 mM) in assay buffer and then prepare a working (500 nM) stock by further diluting 5 ml into 1 ml assay buffer. Aliquot and freeze the remaining stock solution at -20°C.
3.
Add the appropriate amount of Assay Buffer to each well of the 96-well plate (see table below). Blank and cell extract samples are essential for determining cellular activity. To measure non-specific hydrolysis of caspase substrate, preparation of an inhibitor-treated cell extract is recommended. Using a positive-control sample which includes purified caspase enzyme is also recommended. See table on next page.
4.
Allow the 96-well plate to equilibrate to 37°C. The assay may also be performed at room temperature; however, the rate of substrate cleavage will be higher at 37°C.
5.
Add 10 ml of cell extract to the appropriate wells. Do not add cell extract to the blank (control) wells.
6.
Add 20 ml known inhibitor (final concentration 100 nM) or test inhibitor to the appropriate wells.
7.
Pre-incubate the plate at the assay temperature for 10 minutes (or as desired) to allow enzyme/inhibitor interaction.
8.
Start the reaction by addition of 10 ml pNA-conjugated substrate that has been pre-equilibrated to the assay temperature. The final concentration will be 200 mM.
9.
Read the absorbance (A) at 405 nm in a 96-well plate reader. Record data at 1-10 minute intervals for 30 - 120 minutes (as desired, depending on your caspase activity and the amount of protein).
Data Analysis
1.
Plot data as A405 versus time for each sample.
2.
For each sample, determine the initial time period over which the plot of absorbance versus time remains linear, and if there is sufficient change in absorbance (∆A) to obtain an accurate slope. The initial substrate concentration (200 mM) is saturating. For most samples, the rate of substrate cleavage will remain constant for up to 2 hours or more. However, highly active samples can reduce the substrate to sub-saturating levels during the course of the experiment. Therefore, choose the data from the early, linear portion of the curve for use in the slope calculation.
3.
Obtain the slope of the line fitted to the linear portion of the data, using a suitable linear regression program.
4.
Average the slopes of replicate samples.
5.
If the blank has a significant slope, subtract this number from all the samples.
6.
The above data will give a qualitative indication of caspase activity. To quantify the caspase activity in the samples, express as pmol substrate hydrolyzed/min.
7.
Determine the 96-well plate reader conversion factor: A. Prepare a 50 mM stock of pNA-conjugated standard in assay buffer. Add 100 ml to 2 96-well plate wells. B. Determine the average A405 using 100 ml assay buffer as a blank. C. Calculate the conversion factor. This calculation is based on the concentration of pNA in the calibration standard (50 mM). The extinction coefficient for pNA in the assay buffer is 10,000 M-1cm-1. D. Conversion factor (mM/absorbance) = 50 mM ÷ Average A405.
8.
Calculate the activity as pmol substrate hydrolyzed/min: Activity (pmol/min) = slope (absorbance/min) x conversion factor (mM/absorbance) x assay volume (ml).
