Subject: Re: protein concentration

Shaun D. Black SHAUN at JASON.UTHCT.EDU
Wed Sep 27 13:37:14 EST 1995


  Hi all,
  
  Simon Penson replied:
  
  >There is no such thing as an `accurate' protein assay. Each protein
  >behaves too differently in each assay system you choose. So, depending on
  >how much time you want to spend on it, you'll get a rough or not so rough
  
  I totally agree.  Most methods aren't bad, but the standard deviation among
  all proteins is huge.  As a whole, the Biuret-based ones are the best
  (e.g., BCA), but no method is perfect.
  
  >idea. The most accurate protein estimation method is the Kjeldahl total N
  >method, but nobody uses this for routine biochemistry.
  
  I'm not sure that it is the most accurate, but it sure is a pain in the
  neck ;)  .  The approach I like best for calibration of protein assays
  is to run an amino acid analysis.  Do duplicate samples at three times
  of hydrolysis; average and extrapolate the data as appropriate.  Then,
  use the trustworthy residues (not N,Q,W,C,S,T; also not D if done by PTC
  method), and determine the concentration of protein by each residue and
  average the final answer.  Do your protein assay on the same sample that
  you submitted for amino acid analysis.  Compare the concentration values;
  the ratio is your correction factor.  
  
  Best regards to all,  Shaun
  
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    = Shaun D. Black, PhD   | Internet address:     shaun at jason.uthct.edu = 
    = Dept. of Biochemistry | University of Texas Health Center, at Tyler = 
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