Hi,
In order to measure the LDH level in the experimental groups in Pc12 cells, I tried the lactate dehydrogenase activity assay kit (MAK066-sigma). I used one on the brand's page for the protocol, but the results were meaningless. There are some studies that used the same kit but different protocol. I don't know what to do. Is there a protocol that uses this kit or that you can recommend?
I suggest using your LDH Positive Control (or another known high LDH concentration) and serially diluting it to give you 4-6 concentrations (check the kit sensitivity as no point going below it more than once). Test all these in duplicate.
The results from that should tell you where the problems are. e.g does the signal decreases in a linear line? Is there is a large variance between duplicates? That will reveal much information about any method and tell you if it's worth continuing with. If it's not linear around the concentration your expecting, or the variance between duplicates in larger than would give you meaningful results to compare what you want, you need a different method or you need to perform it better. Some of these things do get better with practice as well! Good luck!
I am also using the same kit and having some difficulty with the standard curve. I have followed the instruction in the protocol but the calibration curve is not linear. Could you please share how you were able to get the standard curve?
The kit and worked well but depending on the cell lysis buffer.
PBS with 1% Triton X100 was fine, but with 1% SDS absolutely destroyed the signal.
The final concentrations of detergents in the LDH assay reaction mixes were much less (about 0.03%), so suggest you carefully check that the sample buffers do not interfere with the assay.
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