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
Re: [ccp4bb] Staining Crystals with comassie
Dear Swastik, 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
Re: [ccp4bb] Staining Crystals with comassie
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
Re: [ccp4bb] Staining Crystals with comassie
There are many caveats to using glutaraldehyde on crystals, either for fixing crystals or for staining them. First, I would not hang a 1-2uL drop of 25% glutaraldehyde in the vapour diffusion chamber, but add enough glutaraldehyde into the reservoir to make it 0.5-1.0 % (a 1:25 or 1:50 dilution; a 1% solution is 100 mM). Not only will it be just as effective, the reservoir becomes the control: if the reservoir turns yellow, you have free amines in the system (Tris, ammonium, etc.). Second, the yellow color, which is due to Schiff's base formation, is harder to see in warm light (color temperature, not the temperature of the stage) when you are looking at small or thin crystals. Use cool, white lights (like LEDs). Finally, keeps some buffer around that is suitable for solubilizing the protein. If you are not sure about the color change, just add 10 uL of buffer to the crystals and watch if they dissolve. If they don't when treated with glutaraldehyde, they are protein crystals. As Zhijie said, be careful with handling glutaraldehyde. It is highly volatile and dangerous. If you smell it (and the sweet smell will be obvious), it is fixing you. Keep a waste bottle half-full with 1 M ammonium sulfate, not glycine (too expensive), then just dump any glutaraldehyde waste into it. Cheers, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Oct 16, 2013, at 6:45 AM, Zhijie Li wrote: 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
Re: [ccp4bb] Staining Crystals with comassie
Izit is an aqueous solution of methylene blue, which you can prepare simply and cheaply. Look here: http://www.ysbl.york.ac.uk/ccp4bb/2000/msg00387.html One word of warning: not every crystal stains. I found poking crystal with hair or glass fibre to see if it cracks or breaks is more reliable. Salt crystals react to attacks from glass fibres like rocks (as indeed what they are). From: Swastik Phulera Sent: Tuesday, October 15, 2013 3:59 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Staining Crystals with comassie 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
