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<title>Re: [RASMB] AUC Analysis for a protein without tryptophan</title>
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<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>Hi Arthur et al..<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>Being partly a circular dichroism expert (which means working
mainly in the Far UV) I would say give it ago (we have had lots of success).<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>Arthurs comments are excellent ones..<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>Just be ware of chloride ions as they absorb down there.
Replacement with Fluoride works a treat, just don’t lick you fingers!<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'>Tim Dafforn<o:p></o:p></span></p>
<p class=MsoNormal><span style='font-size:11.0pt;font-family:"Calibri","sans-serif";
color:#1F497D'><o:p> </o:p></span></p>
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<p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span
style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>
rasmb-bounces@rasmb.bbri.org [mailto:rasmb-bounces@rasmb.bbri.org] <b>On Behalf
Of </b>Arthur Rowe<br>
<b>Sent:</b> 26 November 2009 14:17<br>
<b>To:</b> Ritika Sethi; rasmb@rasmb.bbri.org<br>
<b>Subject:</b> Re: [RASMB] AUC Analysis for a protein without tryptophan<o:p></o:p></span></p>
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<p class=MsoNormal><o:p> </o:p></p>
<blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'>
<p class=MsoNormal style='margin-bottom:12.0pt'>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>
<o:p></o:p></p>
</blockquote>
<p class=MsoNormal style='margin-bottom:12.0pt'>--<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>
<o:p></o:p></p>
<p class=MsoNormal><o:p> </o:p></p>
<blockquote style='margin-top:5.0pt;margin-bottom:5.0pt'>
<p class=MsoNormal><o:p> </o:p></p>
<p class=MsoNormal style='margin-bottom:12.0pt'>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><span style='font-size:10.0pt'>_______________________________________________</span></tt><span
style='font-size:10.0pt;font-family:"Courier New"'><br>
<tt>RASMB mailing list</tt><br>
<tt>RASMB@rasmb.bbri.org</tt><br>
<tt>http://rasmb.bbri.org/cgi-bin/mailman/listinfo/rasmb</tt></span><o:p></o:p></p>
</blockquote>
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