About to attempt to blot for Nitrosylation using the Pierce S-Nitrosylation Western blot kit. In the protocol, they suggest to precipiate the protein in 6x volume of acetone for 1 hour. They also note that removal of excess MMTS is require before labeling with iodoTMT reagent and suggest to also use the Zeba spin desalting column. I am wondering if either the column or the acetone work better, if generally they both should be performed for best results, or if there are better priced desalting columns avaible than the one they mention. Any suggestions are appreciated!
In my experience, the best protein precipitation technique before PAGE is the chloroform/methanol method of doi:10.1016/0003-2697(84)90782-6. The protein forms a film, that is easy to isolate and usually dissolves quite well in SDS sample buffer. Thus, recovery of protein is high.
This method will automatically get rid of small molecules, so desalting is not required.
Desalting columns you can make yourself from a disposable syringe, a little glasswool or a plastic fritt (sheets available at BelArts Products) at the bottom and a gel filtration matrix like Sephadex or Sephacryl. The size of the column depends on sample size. The pore size of the gel must be chosen so that the small molecules can enter the pores, but your protein can't. For your purposes, G10 is probably sufficient.
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