BCA Protein Assay and ELISA afterwards - (Jan/17/2013 )
I want to perform Elisa with the brain samples I have. Here's what I've done so far:
-I have taken out mouse brains, took out the regions of interest, weighed them, and homogenated in PBS.
-As it was a last minute decision what regions to analyze and the brains had to be taken out very quickly, I homogenated all regions - regardless of the weight - in 1ml PBS. Then centrifuged and stored the supernatant.
Now my questions are:
-Is it necessary to do BCA assay before doing the Elisa? (I know it is already asked before in a different context, but I am not sure how to proceed).. If yes, based on the results, how should I decide how much (what dilution, what ul) of the samples should I use in Elisa? What is the criteria for it?
- The Elisa kit I have describes in its protocol to homogenate 100mg of tissue in 1ml PBS. in my case it was certainly more than 100mg of tissue I homogenated in 1ml. In this case, would it be more reliable just to follow kit's instructions, and dilute the sample I have according to 100mg:1ml ratio? or would anyone recommend first determining the protein concentration, then deciding based on that?
Thanks a lot in advance. Looking forward to hear any expert recommendations
Homogenization in PBS won't dissolve many of the proteins - you may need to add some detergents and certainly some protease inhibitors to prevent protein degradation.
You should do a BCA as you will have variable amounts of protein in each sample if they are of different weights and as such would get changes in the level of detection (more tissue =greater concentration of protein and hence more likely to detect it). I would recommend that you do straight homogenate and a 1:10 and perhaps 1:100 dilution of the homogenate to ensure that you are inside the reference range for the BCA.
Determine the protein concentration first then dilute to the 100 mg thing.