[RASMB] RE: Reductants, Was: Can 0.25M of NaCl take care of nonideal behavior of a protein?

[John Philo]

Ruby,

I doubt your problems have to do with non-ideality. I assume you meant a
charge of -1.5, not a pI. You never said what protein concentrations you are
using, and salt can never get rid of the excluded volume portion of
nonideality, but I certainly would expect that .25 M of NaCl would suppress
the charge repulsion.

My guess is that your problems are related to the DTT, not nonideality. You
will have variable amounts of DTT oxidation occurring over the course of the
run, causing shifting baseline offsets. See earlier discussions on RASMB
about DTT and other reductants.

[To the group] Speaking of reductants, I noticed recently that Pierce is
selling TCEP immobilized on gel particles. I'm wondering if these could be
put into the cell and basically keep the reductant down at the cell base and
(mostly) out of the light beam. Has anyone tried this? The literature I saw
didn't indicate the particle size, so I don't know whether they would fit
through the loading holes in a 2-channel or external loading 6-channel
centerpiece, but presumably this would at least be possible in a regular
6-channel.

John Philo
Alliance Protein Labs
  -----Original Message-----
  From: rasmb-admin at rasmb-email.bbri.org
[mailto:rasmb-admin at rasmb-email.bbri.org]On Behalf Of Yun-Ru (Ruby) Chen
  Sent: Tuesday, June 04, 2002 9:03 AM
  To: rasmb at rasmb-email.bbri.org
  Subject: [RASMB] Fw: Can 0.25M of NaCl take care of nonideal behavior of a
protein?



  ----- Original Message -----
  From: Yun-Ru (Ruby) Chen
  To: 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
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