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

[Yun-Ru (Ruby) Chen]

Hi, Dr. Laue:
          Thank you for your kindly reply. I have sent out an email describing my conditions, but I don't think it has been posted. Here are the information that I provided in my last email.
        "The concentrations of my sample are as follows: For the ones in Tris/DTT buffer without salt, they are 121, 60, and 30 uM (Abs @280=1, 0.5,
0.25). For those in 0.25M NaCl/ Tris/DTT buffer, they are 79, 36, and 18 uM.(Abs at 280 = 0.65, 0.3 , 0.15). I have rechecked my samples by SDS-PAGE,
they looked fine, at least no other major band present. The speeds I used are 24, 32, and 45K rpm. The sigma factors for those speeds in order are 0.7433,
1.3215, and 2.613. The sigma factor is calculated by the definition (Mb*w^2)/RT.
       About the DTT problems, I have 0.2mM DTT in my samples. The thing is that my protein has only one Cysteine. Therefore, there is no
intramolecular S-S bonds, but perhaps a potential of forming intermolecular S-S bonds."
       The B often comes out negative. Recently, I have some more progress. I have pretty good results from gel exclusion showing this protein is a 1-4-8 (even 10)
association. I rechecked my AUC SE data and found I can fit them all to a 1-4-8 model. The octamer is much less than tetramer, also the tetramer is much less than octamer. When I replace DTT (1 mM) by TCEP (0.5mM) in my buffer (250mM NaCl, 30mM Tris, pH 8), I don't see the octamer anymore. But I still have to confirm this since the loading sample concentration is about 1/3 different. 
       I will check the v bar carefully. We would like to do the sedimentation equilibrium again once we have a better idea from the sizing column. Does anyone know what TCEP concentrations is good for AUC studies? And should I do sedimentation velocity to get more information?
       Dr. Laue, I am very happy to get your reply since I have given a student seminar on AUC and have heard and read about you.
Regards,
Ruby
----- Original Message ----- 
  From: Tom Laue 
  To: Yun-Ru (Ruby) Chen 
  Sent: Friday, June 14, 2002 10:07 AM
  Subject: RE: [RASMB] Fw: Can 0.25M of NaCl take care of nonideal behavior of a protein?


  Hi Ruby,
  Sorry for the tardy reply. I looked over the responses you got and agree with John Philo that it is most likely that your problems stem from baseline offset. A couple of questions: Are you using absorbance optics? At what wavelengths? What concenrations of protein are you using? What rotor speeds did you use?
  When you fit, does B come out negative or positive? If it is negative, then you are looking at association/aggregation. If B is positive, then nonideality is the culprit. The charge on the peptide is not extreme, so I sincerely doubt that nonideality is a problem in either solvent. If you do not get the expected molecular weight when you analyze the data, it may be because of the vbar calculation. In particular, if the peptide has a number of charged residues, the calculated vbar may be to large, leading to estimates of M that are too large. This will be the case even if the net charge is small (similar to what you describe). My experience is that the vbar is about 0.02-0.03 lower if there are ~10-15 charged residues/peptide of this size.
  Best wishes,
  Tom Laue
  University of New Hampshire 
  46 College Road 
  Rudman 379 
  Durham, NH 03824 
  Phone: 603-862-2459 
  FAX:    603-862-0031 
  www.camis.unh.edu 
  www.bitc.unh.edu 

    -----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 12:03 PM
    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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