[RASMB] AUC Analysis for a protein without tryptophan

[Borries Demeler]

> Hi=20
> =20
> I am new to this technique.=20
> 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.
> =20
> Any suggestions please?
> =20
> Thanks
> 

Ritika,

If your protein doesn't absorb at 280, and you have to use buffers or
buffer components that absorb below 280, then interference is still a
good option (provided you have access to an XLI). Although you will
generally see quite a bit of time-invariant noise relative to the
refractive signal at low protein concentration, the time-invariant and
radially invariant noise contributions can be nicely eliminated during
whole boundary fitting, making even low concentration interference data
useful under these conditions.

Keep in mind that in interference experiments, and especially those
performed at low concentration, it is strongly recommended that you 1) use
the dialysate from a thorough dialysis of your protein as your reference,
and 2) meniscus-match the reference and sample sector solution columns,
and 3) use sapphire windows to minimize systematic noise contributions
in your data.

In summary, I concur with Arthur on looking at 220 nm absorbance and 
review the list of low absorbing buffers - we also have an absorbance
profile on the web for several buffers:

http://uslims.uthscsa.edu/cauma/buffer2.php

Good luck, -Borries
---
Borries Demeler, Ph.D.
Associate Professor
The University of Texas Health Science Center at San Antonio
Dept. of Biochemistry, MC 7760
7703 Floyd Curl Drive, San Antonio, Texas 78229-3901
Voice: 210-567-6592, Fax: 210-567-1136, Email: demeler at biochem.uthscsa.edu