Hi Danilo and all,
A little trick for the glutaraldehyde "staining": you can hang a 1-2uL drop
of 25% glutaraldehyde (or the most concentrated stock solution you can find)
besides your crystal drop in the vapour diffusion chamber. The
glutaraldehyde will get into the crystal drop via vapour diffusion. The
color will normally show within 2hrs and become very intense overnight. It
is also a gentle way of crosslinking the crystals
(http://scripts.iucr.org/cgi-bin/paper?wb0066, and
http://hamptonresearch.com/tip_detail.aspx?id=74, ).
!!!!Care should be taken when handling aldehyde concentrates: do not breathe
it, and do not let the vapour get in touch with your eyes. Waste can be
inactivated by concentrated glycine solution.
BTW, the acetic acid in the coommasie blue solution seems unnecessary in a
crystal staining solution. The solution recipe seems to be taken from a gel
staining solution. When staining polyacrylamide gels, the acid (oringinally
HCl) is supposed to denature the proteins so that they do not diffuse in the
gel. The MeOH is for solubilizing the commonly used coommassie R250.
(Another thing: I strongly suggest to substitute the MeOH in PAGE staining
and de-staining solutions with EtOH. EtOH works perfectly fine, without
MeOH's poisonous effect on human. Our staining solution contains 20% EtOH
and 20%HAc.)
For staining crystals, we do not need to add the acetic acid. Also
coommassie G250 is more soluble in water than the R250 by having methyl
groups instead of ethyl groups. 0.5% coommassie G250 can be readily made in
DMSO or 95% EtOH. Then this stock solution can be diluted with water or the
mother liquor 10x-100x for the staining. Many crystals can tolerate up to
10% DMSO.
Zhijie
-----Original Message-----
From: Danilo Belviso
Sent: Wednesday, October 16, 2013 3:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Staining Crystals with comassie
Dear All,
izit dye is a solution containing methylene blue that you could prepare
in your lab. I usually prepare a solution of 0.05%w/v of dye in water
and then I add a volume of dye solution equals to 10% of the volume of
the drop containing the crystal to test. I prefer to add the dye
solution in small portions (if the volume permits) every 2-3h in order
to limit the shock due to the new solution on the crystal. You should
remember that this test is not definitive: the dye is a cationic dye,
that needs of anion counter part to bind the protein. Therefore, the dye
is not able to colour all protein crystals: in addition, colouration is
affect by pH of the crystallization condition, since low pH could
increase the positive charge on the protein reducing its ability to bind
the dye.
You could try also glutaraldehyde as alternative. In order to perform
this test, you should put the crystal into a low ionic strength buffered
solution containing up to 2% glutaraldehyde. In this condition,
formation of Schiff bases with the lysines and N-term residues occurs
and the crystal become a yellow gel, while salt crystals dissolve and
should not be coloured.
To perform comassie crystal staining you should prepare a solution of
1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this
solution as in methylene blue test. However, I rarely use this test
because the use of MeOH and Glacial Acetic Acid causes the crystal
dissolution.
Only a final tip: obviously these tests enable you to distinguish
between protein and salt, however they do not differentiate between
protein in the crystal and protein in solution. For these reason, in
some cases could be difficult to see the crystal colouration due to the
low contrast with the colouration of the solution. Hence, I prefer to
put the crystals to test in a new solution with the same formulation of
the drop where the crystals have grown but without protein and perform
here the dye test that I have chosen. In this way, you can easily see
the colouration of the crystal without background effect.
I hope I've helped you.
Danilo
On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera
<swastik.phul...@gmail.com> wrote:
Dear All,
I am looking for a method to quickly differentiate between salt and
protein crystals. I have been told thats its a popular alternative
to the commercially available izit dye. I would appreciate if some one
would share their comassie crystal staining protocol.
Swastik
