Try again with freshly prepared loading buffer in 8 to 10% of gel. And try to include a positive control sample if possible.

Add DTT to your protein purification buffers since sometimes disulfure bonds could be the answer!!

I agree with Omar, I don't think the proteins were denatured and there may have been some aggregates as well.

use fresh SDS buffer with BME. and boil the sample for 5-15 min

Hi sir, 

What sample you are using here? 

Thanks everyone.. With the fresh reducing sample dye with BME the results are fine now..