Help regarding the function of DTT for SDS-PAGE? - (Aug/29/2012 )
I heat my proteins with 2* SDS sample buffer in 1:1 ratio and run them in SDS-PAGE. I got protein bands. Now when I used DTT with the same sample I didn't get the same bands. top two bands disappeared and instead I got a thick lower band.
I know that DTT reduces disulphide bond and denatures protein more. But I thought its use was optional since adding the SDS-buffer and heating should denature the proteins. I use 5 micro litre of my sample with 5 micro litre of buffer and heat at 100 degree celcius for 5 minutes.
The change in band positions: does that mean the top two bands were the same protein as the lower band? But without DTT wouldn't they give me the band in the same position.
I am so confused and look forward to your replies.
the high molecular weight bands are breaking down to their subunits. the big, low molecular weight band could be all the same but can also be a mixture of equal or near equal molecular weight proteins.