a big problem with INVITROGEN Caspase-3 colorimetrik kit - (Mar/10/2012 )

hi,

i had purchase invitrogen caspase-3 colorimetrik kit for measure apoptosis in c57bl6 mice retina and substantia nigra tissues.when i read the datasheet of kit i realize that,kit was for just cell counting.
i have to make this kit with tissue.i asked this problem with invitrogen tech support but the could not help me.
at datasheet writing at assay procedure 'count cells and pellet 3-5x10^6 cells per sample'
i had searched literature bu i cant find anything about my situation
is the anyone use this kit or another brand?
how will i calculate SN and retina volumes.
shortly how will i do this kit?

thanks for help

The short answer is - you may not be able to use this kit.

The long answer is - It is possible that the kit will still work, but you will have to try it first and play around with the conditions if possible.

A few questions - is this kit for a western blot or for immunohistochemistry? If for westerns, it may well work, however, you would probably be better off just getting an ordinary caspase 3 antibody and doing the westerns for yourself.

thanks for answer
i have to use that kit i have no chance to give it back.
this kit is for ihc.

Having had a quick look at the manual I have 2 comments.
1. This is not a kit for IHC. This kit measures caspase activity by colorimetric assay using a plate reader or spectrophotometer. As far as I can see there is not antibody detection involved.
2. Although it says "count cells and pellet 3-5x10^6 cells per sample"... it also says: "6. Assay protein concentration by any standard method. 7. Dilute each cytosol extract to a concentration of 50-200 μg protein per 50 μL Cell Lysis Buffer (1-4 mg/mL)" .

Now, I am not familiar at all with retina and substantia nigra tissues, nor am I entirely sure what is that you are actually wanting to do. But maybe you could extract protein from these tissues, then measure concentration, and then follow the protocol from step 7: " Dilute each cytosol extract to a concentration of 50-200 μg protein per 50 μL Cell Lysis Buffer (1-4 mg/mL)"





Just my 2 cents.