Hello everyone;
After precipitating the proteins with ammonium sulphate, we performed SDS-PAGE. However, there was a problem with the gel running, we couldn't see any tape. We did dialysis to remove ammonium sulphate. However, no band was again observed in the gel. Even the protein marker wasn't visible. The protein I isolated is
Dear Esra Bedir,
sample and running buffer concentration should be the same. Descrease the polymerization rate. Overlay gels very carefully. The percentage of resolving gel may be increased up to 12.5%. dialysis should be conducted by a 10 kDa cut-off membrane. Please check the concentration of protein in the sample before running the gel. These tips may resolve your problem. If it is not work please ask again.
Thank you very much for your reply. I'll definitely try. I hope I'll make it.
On predicting the pH of pure (NH4)2SO4 aq. sol.; see: https://www.researchgate.net/post/What-is-the-difference-between-using-ammonium-sulfate-and-using-sodium-sulphate-in-protein-ppt-and-seperation
Dear Esra, I would suggest determining the protein content in your sample prior to the SDS-PAGE using Bradford or BCA Assay. You may need to calculate back so that the protein concentration in your sample is around 15-20 microgram protein/well.
Hi Ariestya Arlene Arbita,
Thank you very much, I think your answer must be helpful, and we will do this in our next try.
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