Mystery of SDS gel, working with the protein GB1 (T2Q mutation) - (Mar/31/2015 )

Hey!

I used SDS gel in order to validate the presence of the GB1 protein (6.22kDA) in my sample before and after heating (in order to lysis of the cells) & cleaning with gel filtration column.

It is known that the GB1 domain runs at roughly 13.5kDa despite being only 6.2KDa, so the location is logic.

The mystery is that after the cleaning, there are impurities of allegedly two proteins, a little bit higher and a little bit lower the original concentrated GB1, that don't appear before cleaning.

Could anyone please explain how is it possible?

Thanks,

Tom.

-Tom Aharoni-

a couple of possibilities:

aggregation and decomposition

concentration of contaminants

-mdfenko-

Thank you!

Is there a different kind of gel that could prevent the protein from aggregation and decomposition? native gel maybe?

-Tom Aharoni-

decomposition would occur during handling.

aggregation can occur during handling and/or during heat treatment with sds sample buffer. if it is occurring during the heat treatment then you can incubate at 60-70C instead of 95-100C.

native page will not reduce either and may actually increase aggregation.

-mdfenko-