Regarding to amount of material loaded, I think it may also depend on the buffer. When I did light scattering experiment with membrane protein at angle of 90 degree, I got much better base line with buffer containing DDM than one containing OG. This maybe due to high concentration of OG required I guess. When I increased concentration of protein in OG buffer, it helped increase signal-noise ratio.
In this issue, I don't know if Wyatt column can help reduce the amount of sample. But I doubt if GE 5/150 column has the same resolution with the 10/30 one. When I had mixture of several species, the 10/30 always gave better resolution for me, and this is crucial in obtaining accurate information about oligomerization state and/or absolute molecular weight of the protein. So I think if choosing GE columns for light scattering experiment, I would go for 10/30. Best wishes, Sally. On Tue, Mar 8, 2011 at 7:44 PM, Engin Özkan <[email protected]> wrote: > And as others have said shedding of dextran is a problem with GE columns > (this was confirmed to me by GE people), but after extensive system > equilibration, we do not see a problem significant enough to ever hurt our > light scattering measurements. > > Engin > > On 3/8/11 10:38 AM, Engin Özkan wrote: >> >> On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote: >>> >>> On the other hand, GE Healthcare columns would require a huge amount of >>> material to be loaded. >>> >> What do you mean by a huge amount of material? You would not be using a >> 16/60 column (125 ml column volume) for an analytical experiment. How about >> a Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have >> column volumes at 25 ml and 3 ml, respectively, have great resolution, and >> probably already compatible with your proteins and buffers. >> >> We used to use HPLC columns, but some proteins would never elute from >> these columns. Then we switched to good old Superdex 200 10/300, and it >> works like a charm every time. We inject <100 ul material at concentrations >> around 1 mg/ml (depending on the molecular weight of the protein in >> question). The only issue is we have to run these columns at 0.35 ml/min >> flow rates (instead of the default 0.5 ml/min), since our HPLC has a lot of >> back pressure for FPLC columns. >> >> Best, >> Engin >> > > > -- > Engin Özkan > Post-doctoral Scholar > Howard Hughes Medical Institute > Dept of Molecular and Cellular Physiology > 279 Campus Drive, Beckman Center B173 > Stanford School of Medicine > Stanford, CA 94305 > ph: (650)-498-7111 >
