[RASMB] AUC Analysis for a protein without tryptophan

[Timothy Dafforn]

Hi Arthur et al..
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).
Arthurs comments are excellent ones..
Just be ware of chloride ions as they absorb down there. Replacement with Fluoride works a treat, just don't lick you fingers!
Tim Dafforn

From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On Behalf Of Arthur Rowe
Sent: 26 November 2009 14:17
To: Ritika Sethi; rasmb at rasmb.bbri.org
Subject: Re: [RASMB] AUC Analysis for a protein without tryptophan

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

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Arthur J Rowe
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School of Biosciences
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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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