Re: [ccp4bb] crystal with precipitation
Hi Shivesh I had something like this recently and found that changing the pH stopped the precipitation and the size of the crystals improved. If the crystals are large enough even if there is ppt present I would still try and collect data on them. This is likely not the case for you or you wouldn't be writing in:) hope this helps Rob Dear all I am trying to crystallize a 7kDa protein using MPD as a precipitant at 16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is that the drop is precipitating in one day only and the crystals are coming with precipitation.I have added 5% glycerol and 100mM of Nacl as an additive with the mother liquor to avoid precipitation.But,the precipitation is still there.The volume of the mother liquor is 500 microlt.The drop size is 2+2.I welcome all the suggestions regarding avoiding the precipitation.Thanx is advance. Shivesh kumar -- Robert J.Gruninger B.Sc. PhD Student Department of Chemistry Biochemistry University of Lethbridge, 4401 University Dr. Lethbridge, AB, Canada, T1K 3M4 Phone:(403)329-2746 Fax:(403)329-2057
Re: [ccp4bb] crystal with precipitation
Sorry - this may have been mentioned previously, but have you tried banging in some glycerol (5-10%)? J shivesh kumar [EMAIL PROTECTED] wrote: Dear all I welcome all the suggestions regarding my crystals which is coming with the precipitation.The pI of the protein is 4.2 and the drop precipitates with MPD as low as 30% within 4-5 hrs.I am trying the pH ranging from 3.6-5.2 at 16C.There is no precipitation at 20% of MPD.Also,at high conc,the protein precipitates.The crystals are coming as florets as I mentioned with 2+2.Should I go for higher pH like uptp 8.5 or so.crystals are not growing bigger in size so that I can mount.Should I try changing the temperature.Ican use 4C and 16C.Right now I am keeping plates at 16C.I am concentrating the protein through lyophilization.As soon I pool it for Gel filtration,majority of it get precipitated,not soluble protein.Is there any method to make the protein stable? I appreciate all the informative suggestions.Thanx in advance and thanx everybody who has suggested the various options. Shivesh kumar -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile)
Re: [ccp4bb] crystal with precipitation
I agree to this last suggestion. For one of my protein, I had to add at least 10 % of glycerol in my buffers to keep my protein stable while purifying. For crystallization, I diluted it to 20 % glycerol (vapour diffusion method) which allowed it to be stable in the crystallization drop and eventually leading to crystallization. (well, again it depends from protein to protein). Another thing you could try for bigger crystals is to increase your protein concentration, (if it is stable enough) and set up at 4 C (rather than 16 C). Hope this helps, Manish Sorry - this may have been mentioned previously, but have you tried banging in some glycerol (5-10%)? J shivesh kumar [EMAIL PROTECTED] wrote: Dear all I welcome all the suggestions regarding my crystals which is coming with the precipitation.The pI of the protein is 4.2 and the drop precipitates with MPD as low as 30% within 4-5 hrs.I am trying the pH ranging from 3.6-5.2 at 16C.There is no precipitation at 20% of MPD.Also,at high conc,the protein precipitates.The crystals are coming as florets as I mentioned with 2+2.Should I go for higher pH like uptp 8.5 or so.crystals are not growing bigger in size so that I can mount.Should I try changing the temperature.Ican use 4C and 16C.Right now I am keeping plates at 16C.I am concentrating the protein through lyophilization.As soon I pool it for Gel filtration,majority of it get precipitated,not soluble protein.Is there any method to make the protein stable? I appreciate all the informative suggestions.Thanx in advance and thanx everybody who has suggested the various options. Shivesh kumar -- Professor James Whisstock NHMRC Principal Research Fellow / Monash University Senior Logan fellow Department of Biochemistry and Molecular Biology Monash University, Clayton Campus, PO Box 13d, VIC, 3800, Australia +613 9905 3747 (Phone) +613 9905 4699 (Fax) +61 418 170 585 (Mobile) * Manish B. Shah, PhD. Postdoctoral Fellow Hauptman-Woodward Medical Research Institute 700 Ellicott Street Buffalo, NY 14203. *
[ccp4bb] crystal with precipitation
Dear all I am trying to crystallize a 7kDa protein using MPD as a precipitant at 16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is that the drop is precipitating in one day only and the crystals are coming with precipitation.I have added 5% glycerol and 100mM of Nacl as an additive with the mother liquor to avoid precipitation.But,the precipitation is still there.The volume of the mother liquor is 500 microlt.The drop size is 2+2.I welcome all the suggestions regarding avoiding the precipitation.Thanx is advance. Shivesh kumar
Re: [ccp4bb] crystal with precipitation
Oh, there is do much you can do! Lots of alternatives: 1. Decrease or increase the protein concentration (with a corresponding increase or decrease of precipitant. 2. Use a temperature gradient. 3. Set up in a capillary with liquid-liquid diffusion 4. SEED! #3 or #4 are your best bets, probably. For the capillaries, using Hamilton syringes: Pipet 20 ul protein in a 50 ul capillary. Ease it away from the end. When you are done, make sure you can reach the liquid with the syringe from the other side. Hold your finger on one end. Seal the other end with sticky wax Layer 5 ul water on top. Carefully layer the precipitant (10-30 ul) on top of the water. You might want to use a 4mM glass capillary mounted on a syringe, instead of Hamiltons. Seal the other end. Wait 3 days. Then look. Naturally, you can scale this down using 20 ul capillaries, or even smaller X ray capillaries. But then you can't use syringes to load them, use smaller capillaries. On the upside, you could shoot directly from the experiment. This has worked well for me. Lisa -Original Message- From: CCP4 bulletin board on behalf of shivesh kumar Sent: Wed 8/22/2007 3:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] crystal with precipitation Dear all I am trying to crystallize a 7kDa protein using MPD as a precipitant at 16C.I have got small florets at 55-65% of MPD in 3-4 days.The problem is that the drop is precipitating in one day only and the crystals are coming with precipitation.I have added 5% glycerol and 100mM of Nacl as an additive with the mother liquor to avoid precipitation.But,the precipitation is still there.The volume of the mother liquor is 500 microlt.The drop size is 2+2.I welcome all the suggestions regarding avoiding the precipitation.Thanx is advance. Shivesh kumar
