I'm working with Leismania panamensis and I got a problem using the pellet for western blotting.
I collected a cultive of parasite and washing 3 times with PBS after that I used a DTT and sample buffer and finally heating at 37°C degrees.
I want to know if somebody have a different pre treatment before to do the western blotting?
For Western blot, a sample is better heated up to 100 oC or boiling for 5 minutes in sample buffer. In addition it should be treated with DNAse to reduce viscosity, which is problematic for sample loading and running during electrophoresis. I hope this helpful.
Thanks for answering, I have a few questions more, Is the quantity of the promastigotes important? Did you recommend me a specific number or just a small pellet?? Did you think is important to wash with PBS after I collected the pellet? and How many times? Thanks so much.
It is always a good idea to wash cells in PBS for 2-3 times by suspension and centrifugations to get ride of residual sera in culture media. As far as how many cells are suitable for Western blot, it depends upon many factors, which mainly include 1) quality of antibody; 2) target molecule, how abundant it is; 3)cell growth stages, i.e., log, stationary or purified metacyclic promastigotes; 4) detection methods. You may start with a titration, using 1, 2, 4, 8, 16… million cell each lane in preliminary experiments in your Western blot and then reprobe them with an antibody for loading control such as tubular etc. There are many references in the literature for Western blot. Good luck!
Dear Rita, indeed I agree with Prof. Chaoqun, it appears that he has a good experimental experiences. Here is a paper titled 'ELISA and western blotting for the detection of Hsp70 and Hsp83 antigens of Leishmania donovani", I hope it will be helpful.
Kind regards
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