I want to perform a caspase-3 assay that has not been tested on heart tissue by the manufacturers, but it has been carried out successfully by researchers in a paper that I found (it is usually used for cells). I want to give it a try! The people in the paper homogenize their tissue in 'frozen extraction buffer' prior to performing the assay. They give the concentrations but not the quantities, so I was wondering if anyone has a protocol for extraction buffer that I can please use? Is this the same thing as RIPA buffer? (but if caspases are proteases then I obviously shouldn't add protease inhibitors to the buffer).
Thank you!
Take a look at the attached method from SciGine where the researchers simply blot for Caspase-3 in mouse brains. You DO need protease inhibitors to prevent degradation of the caspases.
http://scigine.com/method.php?ID=77382
Thank you, Karthik. Will the protease inhibitors not then inhibit caspase activity and therefore cause the assay to have a weak signal?
The assay I am doing (not western blot) measures the activity of caspases 3 and 7 in light units. Its a luminescence assay.
Ah, I am sorry. I didn't see that you were doing a luminescent assay for caspase. Take a look at this other scigine method. They use the chemicon kit ... perhaps you can call the company and ask them for the contents of the lysis buffer. Other caspase assays on scigine also use lysis buffer but a majority of then lack protease inhibitors...so perhaps you are correct.
http://scigine.com/method.php?ID=6126
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