u try to run a native gel and see is there any oligomerization. some time what happen, our protein make complex and one of complex subunit is crucial for its stability. when u r heating protein sample, complex is being dissociated. so it might me the reason. u run a native gel and then try to figure out the problem. good luck dear......:p

Hi Gaurav,

For how long do you denature and is this done with your sample tubes closed or open? Also, what sort of lysis buffer and loading buffer are you using? 

I heat the sample for 5 mins at 37 degree Celsius with closed tubes. I do lysis by sonication in lysis buffer containing 10mM imidazole, 300mM NaCl and 10mM TrisCl. I am using 4x loading buffer which contains Beta-Mercaptoethanol, EDTA, Bromophenol blue, Glycerol, TrisCl and SDS.     

Even though you are sonicating, I would suggest perhaps adding some non-ionic detergent to the lysis buffer (triton X or NP-40) to ensure all proteins are remaining in solution.

Also, I'm unsure if you already use this step after lysing by sonication but we centrifuge our lysates at a high speed and 4°C for about 30mins after lysis to pellet the very insoluble fractions/debris.

Thank you Ajeet for your suggestions. 

@Eloise- I do centrifugation for 60 mins at high speed. Adding detergent will not help much as they are mainly involve in dissociating the cell membrane to bring the protein in the supernatant. I am using eluted protein sample from Ni-NTA column. the precipitation might be due to salts present in the sample.