[RASMB] odd equilibrium curves

Joel Mackay j.mackay at biochem.usyd.edu.au
Tue Nov 20 00:19:00 PST 2001


hi all,
Hope i am not bothering everyone with an obvious problem here...

We recorded some equilibrium data this week on a protein of ~ 50 kDa that 
we think is oligomerising. Relevant parameters are:
protein concentration: ~ 10 uM
T: 25 deg
machine: XL-A
buffers: 50 mM HEPES+200 mM NaCl+0.5 mM DTT or phosphate+NaCl+DTT

In these runs we saw a couple of odd things - probably related. Many scans 
had a dip in the 280 near the meniscus so that the curve was clearly 
non-exponential. Some of the scans also had a large 360 absorbance near the 
meniscus. I am not sure what is causing this and would appreciate comments 
from anyone who may have seen a similar thing - or indeed from anyone at all!

Since a picture is worth a thousand words, i have put a couple of traces 
onto a web page:

http://www.biochem.usyd.edu.au/staff/mackay/extras/odd_au.html

Please let me know if i have omitted any important details...

Seems that the DTT is a possible source of problems (and we are about to 
try the run again without the DTT), but i am not sure why it would be 
giving rise to the problem i am seeing here.
cheers
joel


*****************************************************************
Dr Joel Mackay
ARC Research Fellow
Department of Biochemistry, G08
University of Sydney,
Sydney, NSW 2006
Australia
ph +61-2-9351-3906
fax +61-2-9351-4726
WWW: http://www.biochem.usyd.edu.au/staff/mackay/
Sydney Protein Group Website:
http://www.biochem.usyd.edu.au/spg/
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