I am working as a Senior Research Fellow in the Department of Biophysics, Panjab University, Chandigarh, India. I had an opportunity to use and analyze the Caspase 9 Colorimetric Activity Assay Kit from Millipore (Chemicon International). As part of my work, I was in search of a convenient and efficient protocol for evaluation of Caspase 9 activity as I am working on phytochemicals in the area of cancer research. Caspase 9 is an aspartic acid specific protease and is linked to a mitochondrial death pathway of cell apoptosis. Further, stress signals cause release of cytochrome c from mitochondria and activation of apoptosome (apaf1), which in turn leads to cleavage of the proenzyme of caspase 9 and results in its activation. Also, regulation of caspase 9 activation is one of the focuses in discovery for anti-cancer drugs. I tested and evaluated various protocols. I rated them with respect to sensitivity and specificity as well as other aspects such as ease-of-use, hands-on time, the level of complexity of results interpretation and the required skill level of the user.
The chemistry utilized in the Caspase 9 activity Assay Kit involves a labeled substrate, LEHD-p-nitroalanine (pNA), which upon cleavage by active caspase 9, generates chromophore pNA, which can be monitored using a spectrophotometer at 405nm. Further, the kit also contains a caspase 9 inhibitor for verifying the specificity of the substrate.
I found the Caspase 9 Colorimetric Activity Assay Kit instructions to be clear, concise and detailed enough so that anyone skilled in the sciences could perform the assay. Secondly, in the same kit, it is very easy to verify the specificity of the substrate with help of the specific inhibitor which adds to authenticity of observations. Also, the protocol is sensitive enough to allow estimation in a small sample size as an adequate amount of choromophore is available for detection. The only drawback of the Caspase 9 Colorimetric Activity Assay Kit is its cost.
My research involves the use of tissue homogenates. So, prior to dissection, lung tissues were perfused with Tris Buffer, pH 7.4 in order to remove any red blood cell contamination. Tissue homogenates were then made and centrifuged at 10.000 rpm for 15 minutes at 4°C. The resultant supernatant was used in the assay. Then, as directed in the kit instructions, different reagents: caspase 9 substrate solution, active caspase 9 standard, and caspase 9 inhibitor solution, to be used in the assay were prepared. The kit also includes directions for the use of wells on the plate. Accordingly, for the standard wells 100 ul of active caspase standard was pipetted into the standard wells. In the sample wells 90 ul of the samples were added followed by addition of 10 ul of the sample caspase assay buffer. Further, in the inhibitor wells 10 ul of caspase 3 inhibitor solution was added instead of caspase 9 assay buffer. This was followed by addition of 100 ul of substrate to each of the wells as directed in the kit. The intensity of each well was read at excitation 405 nm using a plate reader.
I recommend the Caspase 9 Colorimetric Activity Assay Kit for its technology, reproducibility, sensitivity, and specificity. The instructions were clear, precise and accurate. Further, the reference standard curves helps a lot in the calculation part of the assay.
Department of Biophysics
Panjab University