Reducing agent or not in SDS_PAGE sample buffer? - (Aug/21/2005 )
should we add a reducing agent (DTT or 2-Mercaptoethanol) or not in the SDS_PAGE's sample buffer? The plan is using this gel for Western blot.
Unless there is a specific reason not to, I add DTT to a final concentration of 100mM.
DTT or BME reduces the sample. Unreduced protein might have a different shape than the reduces protein, and this can show up on a WB as a rather large difference in size between reduced and non-reduced protein. The usual molecular markers for WB shows reduced sizes. Also keep in mind that an antibody against a protein is made against either reduced or native protein! The antibody binding sites can be different in these two cases, so you end up with the risk of getting a very weak or no signal if you run your protein in the wrong form.
Wether boiling/heating the sample will lead to complete denaturation, I don't know but it will certainly help.