Amro Here is an extract from our paper, describing a method that is almost infallible, and not too hard to do if you're organized. It can never give false positives and (in the 3 cases we looked at it) only gave false negatives when there was heavy precipitate in the drop.
Best wishes, Patrick Ref: Patrick D. Shaw Stewart, Stefan A. Kolek, Richard A. Briggs, Naomi E. Chayen and Peter F.M. Baldock. 'Getting the most out of the random microseed matrix-screening method in protein crystallization'. Cryst. Growth Des., 2011, 11 (8), pp 3432–3441. On-line athttp://pubs.acs.org/doi/abs/10.1021/cg2001442. In the cases of crystals of the proteins concanavalin A, trypsin, and thaumatin, we used an interesting novel method of making the distinction, which is a modification of the method of Pusey et al.17 We covalently labeled 50 μL aliquots of the proteins with the fluorescent dye DyLight 350 NHS Ester (from Thermo), following the manufacturer’s instructions except that we used higher protein concentrations (30 mg/mL for trypsin and concanavalin A, 36 mg/mL for xylanase). We added 20 nL samples of labeled protein to wells containing putative protein crystals after the crystals had grown. We photographed crystals in a darkroom by illuminating with the UV Pen-280 or with an FL4BLB UV lamp (Luxina), which has a peak wavelength of 370 nm. As shown in Figure 2, crystals fluoresced brightly and were unambiguously identified as protein rather than salt. (The DyLight kits are very easy to use because all resins, columns, etc. are provided. We chose the label that is excited at 350 nm because it is not necessary to use a filter since most cameras have built-in UV filters.) The advantages of the method are (1) since it allows protein to be seen directly, it does not give false positives or negatives (except when the drop contains a lot of precipitate, see below). (2) It cannot interfere with the crystallization process. (3) Labeled protein need only be prepared if crystal identification by other methods fails; (4) even needles and small crystals can be identified. The method does not work well when the drop contains a lot of protein precipitate, which may absorb the labeled protein before it can reach the crystals. Note also that protein sometimes coats salt crystals in crystallization experiments, giving a superficially similar appearance. Such cases can, however, easily be distinguished by comparing UV images with visible light images because the protein coating is outside the salt crystal. (17) Pusey, M.; Forsythe, E.; Achari, A. Methods Mol. Biol. 2008, 426, 377–385. On 11 February 2013 09:37, Ganesh Natrajan <[email protected]> wrote: > > Dear Amro, > > What you could try is this. Make a solution of 0.5 % (w/v) commassie > brilliant blue in 10% (v/v) ethanol in water. Pipet 1 ul of this into your > drop and close the cover slip. If the crystals are protein, they should turn > blue after some time (typically 30 mins). Salt crystals will not turn blue as > they are not stained by commassie. > > You could also try using Hampton's Izit crystal dye for this, but the problem > I have faced with it is that the izit itself crystallizes (gives lovely blue > crystals) under certain buffer conditions. > > cheers > > Ganesh > > > > > > > Hallo my colleagues. > i hope every one doing ok . i did screening since two weeks . i noticed > today this crystals. i don`t know either it salt or protein crystal . my > protein has zero tryptophan so i could distinguish by UV camera. > the condition was conditions: > 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. > > > best regards > Amr > > > > > > > > -- [email protected] Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
