I'm working on Western Blot and was guided to use the Sample Buffer Solution (2ME-) (x4) (Fujifilm Wako). However, Beta-metacaptoethanol (BME) is said to be necessary to cut the disulfide bond in many protocol ( in the absence of BME, proteins with disulfide bonds retain some shape and do not electrophorese consummately by molecular weight) .
So I wonder how important it is in Western Blot in common situation (not special protein) ?
Because some of my senior colleagues told me that they have fine results of WB with sample buffer (2ME-) only (without Beta-metacaptoethanol).
Anyone with experience or know about this please give me some advice. Thank you.
For what purpose you are using the beta- ME. Of course beta- ME helps in dissociation of disulfide bond in protein to denature it. If you want your protein in native form avoid using it.
It depends on the epitope your antibody is supposed to recognise. For linear epitopes, it is often required to break disulfides, although epitopes might be localised on loops that do not involve disulfides. In the latter case, beta-ME can be omitted. However, other proteins eg. tetraspanins must not come in contact with reducing agents as the epitopes are often structural epitopes that are lost upon breaking disulfides, which results in missing signals on a Western!
Sure, depending on your targets (preins), there may be no essential need to use beta-MeEtOH for sample buffer /sample prepation.
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