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Help regarding the function of DTT for SDS-PAGE?


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#1 Bioscientist

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Posted 29 August 2012 - 07:09 PM

Hello

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.

Thanks.

#2 mdfenko

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Posted 29 August 2012 - 08:30 PM

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.
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