[ccp4bb] S-200 buffer-based peak shift?
Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] S-200 buffer-based peak shift?
At 07:23 PM 3/22/2011, Jacob Keller wrote: Dear Crystallographers, I have run my protein-peptide complex several times on a GE S200 10/300 in buffer A (below). Today, to make a crystallization stock, I ran the sample in buffer B, and the peak shifted from a consistent 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that SEC results change as a result of buffer conditions. Could this drastic a shift be due simply to buffer conditions, or could there actually be some buffer/ion-dependent dimerization going on? Anyone have a similar experience? A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) So, it elutes earlier in essentially zero salt. I would bet that the protein is acidic and what you see is a buffer effect. Superdex (and most other gel filtration matrices) carries residual negative charge. So in "zero" salt there will be repulsion between protein and beads, resulting in the protein entering pore less frequently. Hence the earlier elution. I've seen this effect for a couple of monomeric acidic proteins. Chances are, switching to a salt higher than 50 mM will also retard the elution a bit. Typical recommended salt in gel filtration is in 100-200 mM range precisely to suppress ionic interactions. - Dima
Re: [ccp4bb] S-200 buffer-based peak shift?
I was always told that gel filtration resins have a mild ion-exchange character, hence the recommendation to use at least 100mM NaCl in size exclusion buffers. Assuming that it is true, one would expect a protein to stick to the resin in low salt buffers. That is the opposite of what you see (your protein elutes earlier in low salt buffer). That makes me think that you are actually experiencing some sort of oligomerization and/or shape transition. Do you have access to MALS? It would give you a definitive answer. On Tue, Mar 22, 2011 at 8:23 PM, Jacob Keller < j-kell...@fsm.northwestern.edu> wrote: > Dear Crystallographers, > > I have run my protein-peptide complex several times on a GE S200 > 10/300 in buffer A (below). Today, to make a crystallization stock, I > ran the sample in buffer B, and the peak shifted from a consistent > 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that > SEC results change as a result of buffer conditions. Could this > drastic a shift be due simply to buffer conditions, or could there > actually be some buffer/ion-dependent dimerization going on? Anyone > have a similar experience? > > A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) > B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) > > Jacob Keller > > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > -- Mario Sanches Postdoctoral Fellow Samuel Lunenfeld Research Institute Mount Sinai Hospital 600 University Ave Toronto - Ontario Canada M5G 1X5 http://ca.linkedin.com/in/mariosanches
Re: [ccp4bb] S-200 buffer-based peak shift?
Jacob, Some protein can form weak dimer, especially in low salt buffer. AUC can provide a more detailed info about your protein dimerization state. Ray On Mar 22, 2011, at 8:23 PM, Jacob Keller wrote: > Dear Crystallographers, > > I have run my protein-peptide complex several times on a GE S200 > 10/300 in buffer A (below). Today, to make a crystallization stock, I > ran the sample in buffer B, and the peak shifted from a consistent > 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that > SEC results change as a result of buffer conditions. Could this > drastic a shift be due simply to buffer conditions, or could there > actually be some buffer/ion-dependent dimerization going on? Anyone > have a similar experience? > > A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) > B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) > > Jacob Keller > > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > ***
Re: [ccp4bb] S-200 buffer-based peak shift?
Superdex 200 instruction manual suggests a minimal 150mM NaCl is required to prevent binding of protein to the resin. But it seems more to the side of preventing loss of protein instead of misjudging protein size. Nian Huang, Ph.D. UT Southwestern Medical Center On Tue, Mar 22, 2011 at 7:23 PM, Jacob Keller wrote: > Dear Crystallographers, > > I have run my protein-peptide complex several times on a GE S200 > 10/300 in buffer A (below). Today, to make a crystallization stock, I > ran the sample in buffer B, and the peak shifted from a consistent > 16.0 mL to 13.5mL, which would seem to be ~dimer MW, but I know that > SEC results change as a result of buffer conditions. Could this > drastic a shift be due simply to buffer conditions, or could there > actually be some buffer/ion-dependent dimerization going on? Anyone > have a similar experience? > > A: (20mM HEPES, 50mM NaCl, and 5mM CaCl2 pH'd to 8.1 w/ TRIS base) > B: (5mM HEPES, 0mM NaCl, and 1mM CaCl, pH'd to 7.5 w/ TRIS base.) > > Jacob Keller > > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** >
