Use of Urea instead of SDS to denature protein for Western? - (Aug/06/2006 )

Hi Everyone,

I want to try using urea as an alternative chaoptropic agent to denature my microsomal protein before i do a Western blot analysis. Does anyone know what concentration i should use for this? And should I worry that the urea will interact badly with my 5% beta-Mercaptoethanol, which is also in my loading buffer?

thanks,

D

I would use 5M -8M. If you use high purity urea, you should be ok with BME. Some urea contains heavy metals, which speed up the oxidization reaction for BME.

urea gels normally contain 6M urea (i think you can use it in the range of 4-8M).

2-me is no problem.

the only caution: urea enhances separation by charge.