Heat Denaturation without added denaturant (SDS or Urea) - (Jul/28/2015 )
Would heat denaturation (65°C for 15 minutes) completely denature most proteins in the absence of an additional denaturant like SDS? I am performing dot blots and would like to denature my protein to cause a breakdown of structure without the potential foaming that would accompany pulling SDS thru my membrane. Any experience/thoughts?
heat denaturation without sds will most likely lead to aggregates of denatured protein. some proteins are more resistant to heat and some can be made more resistant by adding something to the solution (like ammonium sulfate when purifying atcase) but then you won't be denaturing the protein.
even if it doesn't cause aggregation, it won't open up the molecule like it would with sds.
you might be able to achieve some degree of unfolding (and separate subunits) if you heat in the presence of reducing agent (eg 2-me, dtt).