Whenever I did 2D analysis of my serum sample, it takes too much time to reach its final voltage and also face fluctuation in voltage during IEF.
Please suggest the protocol for IEF program and parameter for serum sample.
Shashank - to me this sounds more like a problem with salt contamination or similar in your sample. Please could you explain your sample prep protocol, including any cleanup and the components you are using in your IEF buffer?
We are not using any de-salting procedure but yes there is some salt concentration that might be a problem,
I would therefore suggest that you run a good acetone precipitation/wash step. Serum proteins should be relatively soluble therefore it is likely that you will be able to get away with a urea based IEF buffer without the need to add thiourea.
In the past we have had problems with thiourea - even though it was a high quality reagent there appeared to be a 'factor' which caused the same sort of problems you suggest. We got past this using cation exchange beads to treat all buffers, but is a pain in the butt. I would therefore suggest that you use as simple a buffer as possible for IEF. Please note, don't over do your ampholytes either - too much of them in the mix can be a problem.
Hope this helps.
Joe
Which is the protein concentration you are using? Sometimes I solved this problem decreasing the amount of protein
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