Reading the instruction manual of BioRad (Catalog number 1652960) is not claer at all the explanation.
The normal solution to have your sample in and rehydrate strips in is 7M Urea, 2M thiourea, 4% CHAPS and 1% ampholytes. I hate CHAPS because it can interfere with MS if not properly removed. Instead I use 1% C7BzO, which is a better surfactant than CHAPS. The ampholytes are optional as well and will depend on the sample. Ampholytes can help keep proteins in solution during focusing.
You could use 8M urea instead of the 7M/2M and probably see little difference, but numerous papers from the late 90's showed that the 7M/2M was more chaotropic and solubilised more proteins than urea alone.
The normal solution to have your sample in and rehydrate strips in is 7M Urea, 2M thiourea, 4% CHAPS and 1% ampholytes. I hate CHAPS because it can interfere with MS if not properly removed. Instead I use 1% C7BzO, which is a better surfactant than CHAPS. The ampholytes are optional as well and will depend on the sample. Ampholytes can help keep proteins in solution during focusing.
You could use 8M urea instead of the 7M/2M and probably see little difference, but numerous papers from the late 90's showed that the 7M/2M was more chaotropic and solubilised more proteins than urea alone.
Hi Florencia,
I agree with Matthew regarding the concentration of Urea and Tiourea, and ampholytes (3-10).
If you are working with total extracts, the 7M/2M ratio suggested from Thierry Rabilloud (http://www.ncbi.nlm.nih.gov/pubmed/?term=Thierry+Rabilloud) have demonstrated to give better dissolution of of a wide range of proteins in complex mixture.
Moreover, if do not have C7BzO, the sulfobeteine ASB14 is a valuable alternative to CHAPS.
Some information in this regard can you find hier:
http://arxiv.org/ftp/arxiv/papers/0904/0904.0685.pdf
cheers
Ignazio
Hi Matthew & Ignazio!
Thank you very much for your contributions. I hope to do it.
Regards
In my experience C7BzO is a good "all-round" surfactant whereas ASB-14 seems to work better on solubilising membrane proteins. I have no empirical evidence to support this though.
Dear Florencia
You have two choices:
1- Use 7M urea and 2M thiourea 4% CHAPS and 1% ampholytes
or
2- Use 9M urea 4% CHAPS and 1% ampholytes
The results obtained by thiourea are more better.
i had use 9M urea, 4% NP-40, 2% ampholytes, 1% DTT for sample buffer preparation
for sample ouverlaying solution u can use 8M urea.
I recently switched from 7M Urea/ 2M Thiourea to 8M Urea and have seen changes in my focusing pattern. I will likely be switching back. However will addition of ampholytes cause any significant change in focusing pattern?
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