denaturating protein electrophoresis - (Jan/23/2006 )
Hi
I was wondering if DTT as a denaturation agent for loading protein lysate on the gel is stonger than BME...could you suggest me a strong way to denaturate my protein, the usual 100mM DTT leaves most of it stuck at the top of the gel...
thanks in advance for the help
Francesca
DTT is not a denaturant, it is a reducing agent.
If you proteins are stuck in the gel well that means they are not solubilizing.
What is the %SDS you suspend your samples in? Maybe it is not high enough.
If you proteins are stuck in the gel well that means they are not solubilizing.
What is the %SDS you suspend your samples in? Maybe it is not high enough.
So then what is a denaturant? BME?
Is SDS a denaturant?
our % of SDS is .4% final in each sample.
sorry we know DTT is a reducing agent and not a denaturant.
We are able to see some bands around 64KDa but we don't see them at the expected size (19KDa)..so the protein lysate is running fine but the protein we are looking for is not appearent, the lysate should contain this protein but maybe we should lysis the cells with some stronger buffer. Do you have any other suggestion?
thanks for the help
Francesca and Geryl
Up your SDS to 2-4%.
You also might want to add some triton x-100 to the lysing buffer.
Boiling denatures the proteins.
BME is like DTT in that it is a reducing agent.
the DTT molecule is like two BME molecules attached together....