During SDS-PAGE, I am preparing the protein samples, I denature the protein via boiling alongwith loading dye for 5 min. But after boiling protein samples are getting precipitated, I had never faced this problem earlier. Can you please suggest the solution?
Demnatured proteins may expose hydrophobic regions leading to aggregation of proteins.
Look at the total protein concentration, it may be too high to keep your proteins solubilised. If that is OK, then look at the concentrations of your buffers, loading dye etc. Then rerun the experiments with fresh reagents.
First of all, how did you come to know that only protein component is getting precipitated ?? it may or may not be SDS as well . Though, You take the supernatant and check the protein content and compare with fresh samples before adding the sample in the sample buffer. Sometime isoelectric pH of the protein sample becoz of any reason like buffer pH or any other ingredient while heating may also cause precipitation.
Thanks alot for your valuable inputs Mr. Rao, Andrew and Naresh.
As Mr Naresh asked, when I prepared denatured sample, i tuk the supernantent and quantify the protein concentration with the freshly isolated sample but there was almost no protein in denatured sample. that's where I got the doubt that my protein is getting precipitated. As the concentration of isolated protein is very high. I will dilute the sample and check it again. I hope it works. Once again thanks alot. Let me check and then will add the inputs.
Cheersss!!!!!!!!!!!!!
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