<HTML>
<HEAD>
<TITLE>Re: [RASMB] AUC Analysis for a protein without tryptophan</TITLE>
</HEAD>
<BODY>
<BLOCKQUOTE>Hi Ritika (and all)<BR>
<BR>
What does the u/v spectrum look like? And is there no tyrosine present? Whatever, you can get a massive gain in sensitivity by going down into the far u/v, so long as you take care to avoid the presence of things such as EDTA, CyDTA, DTT and similar. Good clean windows (use near-boiling neutricon), quality lens tissue to polish, you can get down to near 200 nm, but often you will find a peak in absorption around 220 nm. And don't forget - using modern software you can get away with absorption levels so low (e.g. plateau at 0.05 OD units or less) that you can hardly see the trace above baseline. I have published papers with everything done in the far u/v. It's easy.<BR>
<BR>
As regards the buffers which are suitable, I have a little document giving values for absorption at 200 nm & 220 nm for a range of commonly used buffers. The 'pick of the bunch' are:<BR>
<BR>
CHES<BR>
glycine<BR>
MES<BR>
MOPS<BR>
Phosphate<BR>
TAPS<BR>
TES<BR>
<BR>
all of which have (at 10 mM) an OD <0.1 at 220 nm, and generally <0.5 at 200 nm. BUT - serious amounts of neutral salt can lead to an increase in OD, even though the salt may have little absorption of its own. Glycine buffers are bas about that issue.<BR>
<BR>
Additionally, acetate, bicine, cacodylate, glutamate, pyrophosphate & TRIS are OK at 220 nm (i.e. OD <0.3).<BR>
<BR>
All best wishes<BR>
<BR>
Arthur<BR>
<BR>
<BR>
</BLOCKQUOTE>--<BR>
*******************************************************<BR>
Arthur J Rowe<BR>
Professor of Biomolecular Technology<BR>
NCMH Business Centre<BR>
University of Nottingham<BR>
School of Biosciences<BR>
Sutton Bonington<BR>
Leicestershire LE12 5RD UK<BR>
<BR>
Tel: +44 (0)115 951 6156<BR>
+44 (0)116 271 4502<BR>
Fax: +44 (0)115 951 6157<BR>
email: arthur.rowe@nottingham.ac.uk<BR>
Web: www.nottingham.ac.uk/ncmh/business<BR>
*******************************************************<BR>
<BR>
<BR>
<BLOCKQUOTE><BR>
</BLOCKQUOTE><BR>
<BLOCKQUOTE>Hi <BR>
<BR>
I am new to this technique. <BR>
My protein precipitates in most of the buffers and one can't go beyond a certain (very low) concentration. I was hoping to screen this protein against different buffers and see micro-aggregates on the AUC and then try different additives to remove the microaggregates. However, I believe its impossible to do so if the protein is without tryptophan.<BR>
<BR>
Any suggestions please?<BR>
<BR>
Thanks<BR>
<BR>
<TT>_______________________________________________<BR>
RASMB mailing list<BR>
RASMB@rasmb.bbri.org<BR>
http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb<BR>
<BR>
</TT></BLOCKQUOTE><TT><BR>
</TT>
<br/>
<p>
This message has been checked for viruses but the contents of an attachment
may still contain software viruses which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.
</p></BODY>
<br/>
<p>
This message has been checked for viruses but the contents of an attachment
may still contain software viruses, which could damage your computer system:
you are advised to perform your own checks. Email communications with the
University of Nottingham may be monitored as permitted by UK legislation.
</p>
</HTML>