[RASMB] AUC Analysis for a protein without tryptophan

[Arthur Rowe]

Hi Ritika (and all)

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.

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:

CHES
glycine
MES
MOPS
Phosphate
TAPS
TES

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.

Additionally, acetate, bicine, cacodylate, glutamate, pyrophosphate & TRIS
are OK at 220 nm (i.e. OD <0.3).

All best wishes

Arthur


--
*******************************************************
Arthur J Rowe
Professor of Biomolecular Technology
NCMH Business Centre
University of Nottingham
School of Biosciences
Sutton Bonington
Leicestershire LE12 5RD   UK

Tel:        +44 (0)115 951 6156
           +44 (0)116 271 4502
Fax:        +44 (0)115 951 6157
email:      arthur.rowe at nottingham.ac.uk
Web:        www.nottingham.ac.uk/ncmh/business
*******************************************************




Hi 

I am new to this technique.
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.

Any suggestions please?

Thanks

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