In our lab we do DLS on the SEC fractions and only set up those that are monodisperse :-)
On Mon, Jul 1, 2013 at 7:37 PM, El Arnaout, Toufic < elarnao...@biochem.wustl.edu> wrote: > 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 [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. > Berry [ber...@upstate.edu] > Sent: Saturday, June 29, 2013 9:34 PM > To: CCP4BB@JISCMAIL.AC.UK > 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: CCP4BB@JISCMAIL.AC.UK > > 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 > > >
