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 <[log in to unmask]> 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
>
|