How much protein needed for sequencing

[Chaan Gurer]

In article <3u1o5g$is0 at azure.acsu.buffalo.edu>, xinguo at acsu.buffalo.edu
(Xin Guo) wrote:

> Dear bionetters:
> 
> I have a protein that is transiently expressed in HeLa cells. I want to 
> immunoprecipitate it and then analyse the phosphorylation site. I am
> concerning the amount of the protein for microsequencing. I will digest
> the protein with trypsin and get the phosphorylated fragments from SDS-
> PAGE and then microsequence the fragments. My question is that how much
> the peptide is required for the sequencing. 

It might be a good idea to read this paper:
      Merrick, Alex; Electrophoresis 1994, 15, 735-745
He considers the lower limit for protein sequencing at about 20pmole. The 
lower limit would be 1 ug for a 50kDa protein. I hope this helps.
Chaan Gurer
UIUC
Cell and Structural Biology


BTW, should I digest the 
> immunoprecipitated complex directly or digest gel-purified protein? 
> 
> I am new in protein chemistry. Any suggestions are greatly appreciated.
> 
> Xin
> Dept. Pharmacology
> SUNY at Buffalo
> -- 
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