Hi there,
Parasites are separated from RBCs using saponin lysis buffer and then parasite pellet is resuspended in RIPA buffer or any cell lysis buffer with protease inhibitors followed by sonication. Lysate is centrifuged at maximum g force at 4C for 15 mins. Protein concentration of the supernatant is estimated and 50 micrograms is resolved on SDS PAGE for Western detection.
I do see a positive signal indicating the presence of my protein of interest. However very hazy (Probed with either anti-protein or commercial antibody). I have attached the image for reference. Could anyone see any reason for such observation? any possible solutions ??
Thank you
Regards
Prasoona.
The resolution of the bands is poor. Partly, this may be due to overloading the lane. Partly it may be due to insufficient solubilization of the proteins in the sample (too much protein for the amount of detergent). There is also some lateral spreading, which is caused by the sample having a higher salt concentration than the adjacent lanes.
Dear Dr. Shapiro,
Thanks for the kind reply. I did try lower concentrations of lysates which also resulted in exactly similar result. In general handling parasite lysates has been a problem.
Regards
Prasoona.
Do you notice any viscocity in your sample?? It could be that the released DNA is impairing the migration of protein out of the well and might account for the fact that you see the same results at a lower sample concentration. Sonicating the samples should be adequate but you could either treat with a DNAse or, if you have enough sample, perhaps simply sheer the DNA mechinically with syringe and fine bore needle.....try not to generate too much frothing though!!
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