[RASMB] Fw: Can 0.25M of NaCl take care of nonideal behavior of a protein?

[H. Olin Spivey]

I agree with John Philo.  Although tri-n-butyl phosphine is not 
perfect in this regard, we find it much superior to DTT or beta 
mercaptoethanol for A280 measurements.

Olin
__________________________________


At 12:02 PM -0400 6/4/02, Yun-Ru \(Ruby\) Chen wrote:
>
>----- Original Message -----
>From: <mailto:ychen5 at unity.ncsu.edu>Yun-Ru (Ruby) Chen
>To: <mailto:rasmb at rasmb-email.bbri.org>rasmb at rasmb-email.bbri.org
>Sent: Friday, May 31, 2002 2:37 PM
>Subject: Can 0.25M of NaCl take care of nonideal behavior of a protein?
>
>Dear RASMB members:
>               I have currently run into problems by analyzing my 
>data from sedimentation equilibrium studies. I am very confused and 
>frustrated about it.
>I hope if any of you can give me some ideas. I am working on a 10KDa 
>protein domain which is suppose to be a helical bundle. The pI of 
>the protein is calculated to be about -1.5 at pH 8. I have ran the 
>samples in two different buffers, one in 30 mM Tris, pH8, and one in 
>Tris with 250 mM NaCl. Both buffers contain 0.2mM DTT and are at pH 
>8. However, we cannot fit all the data to a single assembly model. I 
>have tried to put in different guesses of "B" values for 
>nonideality, but the fits are even worse. Moreover, the data from 
>the salt buffers look better then the one without. Therefore, does 
>that mean 0.25M NaCl is not enough or there is something else that I 
>should be aware of? My protein is much less soluble in the presence 
>of NaCl, so it is a bit hard for me to increase to salt 
>concentrations.
>Thanks very much if you can share your idea with me.
>
>Sincerely,
>Ruby
>
>Yun-Ru (Ruby) Chen
>Ph.D. candidate
>Department of Structural and Molecular Biochemistry
>North Carolina State University


-- 
H. Olin Spivey                                Phone: (405) 744-6192
Dept. Biochem. & Molec. Biology               Fax:   (405) 744-7799
246 NRC                                       Email: 
OSpivey at Biochem.Okstate.Edu
Oklahoma State University
Stillwater, OK 74078-3035