Caspase-3 Cellular Activity Assay Kit

Caspase-3 Cellular Activity Assay Kit

Stock Code: 3586489
Manufacturer Part No: 235419-1KIT
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Analysis Note


1. Plot data as A405 or arbitrary fluorescence units (AFU) versus time for each sample.2. For each sample, determine the length of the initial time period over which the plot of absorbance (or AFU) vs. time remains linear, and there is sufficient change in absorbance (or change in AFU) to obtain an accurate slope. The initial substrate concentration (200 µM DEVD-pNA) is saturating. For many samples the rate of DEVD-pNA cleavage will remain constant for at least 2 h. Highly active samples, however, can reduce the substrate concentration to sub-saturating levels in substantially less time. In such cases, choose data from the earlier, linear portion of the time course for use in the slope calculation of Step 3. (For an example, see the "Etop.-4" data in Fig. 2). Since the AMC substrate is used at a sub-saturating concentration (30 µM), extra care must be taken in choosing data that lies within the linear portion of the reaction progress curve.3. Obtain the slope of the line, fitted to the linear portion of the data, using an appropriate linear regression program.4. Average the slopes of replicate samples.DATA ANAYLSIS (in terms of substrate conversion)5. If the blank has a significant slope, subtract this number from all samples. Under normal conditions this will not be necessary since the slope will be nearly zero.6. Specific Activity Calculationsa) To normalize activities with respect to cell number, divide the values calculated in step 6 by the number of cells suspended in 10 µl of lysis buffer. (see Experimental Methods, steps 1-3):Specific Activity (pmol/min/number of cells) = activity (pmol/min)/number of cells per wellb) To calculate specific activities with respect to total protein, determine the protein content of each cell extract. Divide activities by the protein content of the corresponding sample (see Figures 2 and 3):Specific Activity = pmol/min/mg proteinNote: The cell lysis buffer is compatible with Coomassie dye binding (Bradford) and bicinchoninic acid (BCA) based protein assays under appropriate conditions. For the BCA assay, cell extract samples must be diluted ≥10-fold in water. Increased backgrounds may be exhibited with the BCA assays due to the increased DTT concentration. Cell extracts diluted >10-fold with water are considered compatible. Be sure to use cell lysis buffer in the standards and maintain equal amounts in all samples.Note regarding AMC calibration standard: The exact AMC concentration range that will be useful for preparing a standard curve will vary depending on the fluorimeter model, the gain setting, and the exact excitation and emission wavelengths used. The AMC standard, as provided (30 µM), may yield off-scale readings in some cases. We recommend diluting some of the standard to a relatively low concentration with Assay Buffer (0.5 or 1.0 µM) and then measuring the fluorescence of 100 µl. The estimate of AFU/µM obtained with this measurement, together with the observed range of values obtained in the enzyme assays, can then be used to plan an appropriate series of dilutions for a standard curve.


Components


Human Recombinant Caspase-3, Cell Lysis Buffer, a DEVD-pNA Colorimetric Substrate, Ac-DEVD-AMC Fluorometric Substrate, Calibration Standard (p

Quality Level100
ManufacturerSIGMA-ALDRICH
Storage Temp.−70°C

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