Precise protein concentration

Ian McFarlane i.mcfarlane at icrf.icnet.uk
Wed Jan 6 12:03:41 EST 1999


Try a Lowry assay in the prescence of SDS. Also if you want an amino acid
analysis you can just pellet your protein with acetone it doesn't have to
be in soln.

Ian Mc






In article <36937EC2.BD70B3FA at biochem.purdue.edu>, Lena Zaitseva
<zaitseva at biochem.purdue.edu> wrote:

> --------------B5962DAB68528C118428EC82
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> =A0=A0=A0=A0 Hi,
> =A0=A0=A0=A0 We purified a membrane protein in the presence of non-ionic
> detergent. Now we need to calculate precise protein concentration. It
> appeared to be a real problem.
> =A0=A0=A0=A0 Bradford method doesn't work at all. BCA method looks better=
> , but
> with BSA as control it doesn't give us reliable results (there is no
> linear dependence of OD from concentration of our protein). For
> UV-method it would be nice to unfold=A0 protein to get the maximum
> absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein
> aggregation. We tried also make quantitative amino-acid analysis of the
> protein, but=A0 presence of high salt concentration (1.5 M) and glycerol
> in the sample buffer is a problem in that case.
> =A0=A0=A0=A0 Do you have any idea about that?
> =A0=A0=A0=A0 Thanks for your suggestions.
> =A0=A0=A0=A0 Lena.
> 
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> Content-Type: text/html; charset=us-ascii
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> <HTML>
> &nbsp;&nbsp;&nbsp;&nbsp; Hi,
> <BR>&nbsp;&nbsp;&nbsp;&nbsp; We purified a membrane protein in the presence
> of non-ionic detergent. Now we need to calculate <FONT SIZE=+1>precise</FONT>
> protein concentration. It appeared to be a real problem.
> <BR>&nbsp;&nbsp;&nbsp;&nbsp; Bradford method doesn't work at all. BCA method
> looks better, but with BSA as control it doesn't give us reliable results
> (there is no linear dependence of OD from concentration of our protein).
> For UV-method it would be nice to unfold&nbsp; protein to get the maximum
> absorbency at 280 nm. However, guanidine-HCl and SDS lead to the protein
> aggregation. We tried also make quantitative amino-acid analysis of the
> protein, but&nbsp; presence of high salt concentration (1.5 M) and glycerol
> in the sample buffer is a problem in that case.
> <BR>&nbsp;&nbsp;&nbsp;&nbsp; Do you have any idea about that?
> <BR>&nbsp;&nbsp;&nbsp;&nbsp; Thanks for your suggestions.
> <BR>&nbsp;&nbsp;&nbsp;&nbsp; Lena.</HTML>
> 
> --------------B5962DAB68528C118428EC82--



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