We have human samples. We have used RIPA buffer to extract protein and to perform western-blot analysis. I'm interested to measure AMPK activity in these samples but I don't have froze muscle.
I have no specific experience with AMP kinase assay, so I dont know whether any other buffer would better preserve AMP kinase activity. But generally for kinase assays, SDS and salt concn in the lysis buffer could be the main problem. If you have the possibility to dilute it 5-10 times in the reaction buffer, it should be OK.
There are many RIPA recipes around. Often they contain about 0,1% SDS and/or 1% Sodium Deoxycholate, both of which are ionic detergents which tend to negatively affect binding to the ELISA plate.
In my experience ELISA works with such RIPA buffers when diluted in nomal ELISA-buffer 1:5 to 1:10.
The SDS in the RIPA buffer will likely cause at least partial denaturation of the kinase rendering the activity measurements unreliable. Can you try other lysis agents that include only non-ionic detergents (these may still be problematic). Hypotonic lysis might be the way to go. If the extracts available were all made with RIPA, then dilution is your best bet.
Hi, Thanks all for your constructives answers.
In my RIPA buffer I have 0.1% of SDS, 0.5% of Sodium deoxycholate and 150mM NaCl. Do you think I can try an ELISA ? It cost a lot and we don't have a lot of money in my lab...
Finance is always an issue now a days in life-science...
If it was only ELISA, just diluting and using would have been completely risk free. Only problem here is how much activity could have been lost due to the effect of these strong detergents. I know protocols using RIPA buffer to immunoprecipitate kinases for assay (at least for some kinases like Src). So I hope that the components will not have too drastic effects.
https://www.scbt.com/protocols/Immune%20Complex%20Protein%20Kinase%20Assays.pdf
If you have no choice, I would suggest to include good controls- for example some solid positive and negative controls (starved and stimulated cell culture lysates; inhibitor treated cell lysates etc) prepared in the same lysis buffer. ( In addition to dilution in reaction buffer). Good Luck
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA