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 <[log in to unmask]> 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
>
>
>
>
>
>
>
>



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