If the SDS-PAGE sample can not be heated... - Help (Sep/23/2006 )
Hi,
Because of the weird characteristics of the protein I want to detect, my samples for SDS-PAGE gel can not be heated. What I have been doing is add 8M urea into SDS sample buffer.
Recently I have been seeing extra bands, smear or complete dark in the lanes. Wondering if it is because the GST-fusion samples are not completely denatured.
Does anyone have any idea 8M urea in SDS but no heating would complete denature the protein, especially GST fusions, since GST is so easy to dimerize? I always have 1mM DTT in the lysis buffer, and 10% beta-ME in the 2x SDS sample buffer.
Thanks a lot.
why you cannot heat your protein? its not going to break 
Hi,
Because of the weird characteristics of the protein I want to detect, my samples for SDS-PAGE gel can not be heated. What I have been doing is add 8M urea into SDS sample buffer.
Recently I have been seeing extra bands, smear or complete dark in the lanes. Wondering if it is because the GST-fusion samples are not completely denatured.
Does anyone have any idea 8M urea in SDS but no heating would complete denature the protein, especially GST fusions, since GST is so easy to dimerize? I always have 1mM DTT in the lysis buffer, and 10% beta-ME in the 2x SDS sample buffer.
Thanks a lot.
Well, it is known that when it is heated, the protein will precipitate in SDS and therefore trap between stacking gel and resolving gel. It is therefore people in the filed usually use 8M urea to replace heating.
Hi,
Because of the weird characteristics of the protein I want to detect, my samples for SDS-PAGE gel can not be heated. What I have been doing is add 8M urea into SDS sample buffer.
Recently I have been seeing extra bands, smear or complete dark in the lanes. Wondering if it is because the GST-fusion samples are not completely denatured.
Does anyone have any idea 8M urea in SDS but no heating would complete denature the protein, especially GST fusions, since GST is so easy to dimerize? I always have 1mM DTT in the lysis buffer, and 10% beta-ME in the 2x SDS sample buffer.
Thanks a lot.
8M urea is very strong for breaking disulfide-bridges.
However, DTT also, but especially with heating. It could be that this is the reason for using 8M urea, it might work better than DTT unheated, but I'm not sure.
This is quoted from wikipedia (searched on DTT)
DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. However, even DTT cannot reduce buried (solvent-inaccessible) disulfide bonds, so reduction of disulfide bonds is usually carried out under denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride or 8 M urea).
http://en.wikipedia.org/wiki/Dithiothreitol
this means that your smears are probably being caused by overloading of the gel.
what method are you using to stain your gel?
if you are using silver then you may want to stain with coomassie.