Hello Peter,
In addition to the great comments/details, please check the following points I have now in mind.. since you want to relate the size exclusion peak/profile to the crystallization:
- occasionnaly, some perfect peaks that you might think are homogeneous actually correspond to a sample of hetergeneous protein (maybe the target protein will still crystallize, but problems happen during crystal optimization, or/and observing missing electronic density of the N- or C-terminus for example). It might also be reflected on the specific activity (btw if the protein you have is easy to assay, you could check the activity from different fractions if you think broad peak = problem). In some occasions, analyzing fractions from a "perfect" peak shows on SDS-PAGE a double band or sometimes far bands (I won't comment on oligomerization in this case).
- not all proteins from good SE chromatograms crystallize...
- some people only collect fractions from the centre of the peak (or for example they measure the A280 max, divide it by two, draw an horizontal line at that value, and collect the fractions/projection between where the line crosses the peak from both sides.. If you add one fraction before or after, the protein might not crystallize anymore.
- from the literature, I have seen many shapes of chromatograms: perfect, bleeding, skewed (tail) to the right or left, little broad, etc.. which resulted in diffraction quality crystals.
- re-running a protein sample (that crystallizes after the first SEC) for a second SEC might cause the protein not to crystallize anymore (for example membrane proteins might lose lipids etc).
- for proteins that are subjected to SEC straight after Ni-NTA, and which have a perfect peak shape: a 4-16 hours delay before injection might show the aggregation effect. A peak shoulder will form, which you might not have seen if the sample was directly injected. The point is: maybe what you incubate for crystallization or work on after SEC might not be anymore that protein with the beautiful peak you had. Using different additives/chemicals/mutations may help in making the protein more stable/thermostable. You can check previous publications of Dr Tate CG, GPCRs for example. This is also a nice paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111809.
Regards
toufic el arnaout
School of Medicine - 660 S Euclid Ave
Washington University in St. Louis
St Louis, MO 63110, USA
________________________________________
From: CCP4 bulletin board [[log in to unmask]] on behalf of Edward A. Berry [[log in to unmask]]
Sent: Saturday, June 29, 2013 9:34 PM
To: [log in to unmask]
Subject: Re: [ccp4bb] off topic: good peak on gel filtration
Thanks- guess I'm old-fashioned, using low-pressure columns.
So apparently theoretical plates are still calculated, and have
improved a lot- 25000/m is HETP .04 mm, way better than the
figure I mentioned. (TP per dollar not so much.)
No more sour grapes from me-
eab
Zhijie Li wrote:
> Hi Ed,
>
> I guess by "24mL SD200" Peter meant the Superdex 200 10/300 column, which
> most of us should be quite familiar with. According to GE healthcare, a new
> Superdex 200 10/300 GL column should have TP >25000/m. For comparison, a new
> Superdex200 16/600 PG, which uses bigger beads, has TP >13000/m. The TP
> difference of the two should be mainly caused by the different resin sizes.
> Of course in reality columns change over time and in cases like Peter's, it
> might be a good idea to test the performance of the column before drawing a
> conclusion. When we are concerned about resolution of a column, we load a
> standard sample and calculate the TP based on the peak shape. As I remember,
> GE healthcare's SEC manuals has recommended procedures on TP determination.
>
> Zhijie
>
>
> -----Original Message-----
> From: Edward A. Berry
> Sent: Saturday, June 29, 2013 7:43 PM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] off topic: good peak on gel filtration
>
> Peter Hsu wrote:
>> Hi all,
>>
>> I've generally always thought as long as the peak was symmetrical and not
>> too broad would suggest a good sample. However, looking at my previous
>> runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or
>> slightly broader peaks with about 3mL (all symmetrical peaks, roughly
>> similar amounts loaded on the columns). I'm curious to see what people's
>> views are as far as what constitutes a broad peak and how much that can
>> end up affecting crystallization of the sample.
>>
>> Thanks for any responses.
>>
>> Peter
>>
> The width itself may not be a good indicator unless its always the same
> protein- in general a molecule that elutes later
> will have a broader peak.
> Supposing that each time a molecule diffuses into the stationary phase it
> resides there for a certain time on the
> average, then the extra retention time is proportional to that time, times
> the number of times it enters stationary
> phase (N, "theoretical plates"). The variance in elution time is
> proportional to the square root of N (like standard
> error of the mean) and the dwell time. This gives sigma/(retention time) =
> 1/sqrt(N). If N is the same for all
> molecules, the criterion to look at is peak width divided by retention time.
> If it varies (the reason some molecules
> elute slower is not just that they stay in the stationary phase longer, but
> also they enter more often; k-on as well as
> k-off) that would still be better than just peak width. People don't talk
> about theoretical plats and HTEP much any
> more, perhaps because the driving force in chromatography is HPLC and FPLC,
> and fast chromatography is antithetical to
> good resolution?
>
> However I'm not familiar with this column and can't advise. You can
> calculate N more exactly (see wikipedia "van Deemter
> equation") as 8*ln(2)*square of (elution volume over width at half height),
> divide length of column by that to get HETP,
> and compare with values like .7 mm reported for resins like ultragel A at
> optimum (very slow) flow rate.
>
> eab
>
|