Re: [ccp4bb] zinc with HEPES
Order of addition when making the solutions is often important to dilute the least soluble component rapidly. Zn acetate is the most soluble Zn salt. Put that in your tube first and then dilute it with your largets volume (probably either water) and then add the other ingredients, consider vortexing in between additions. Jim On Fri, May 11, 2012 at 9:26 AM, Rajesh Kumar wrote: > Dear All, > > This question sounds simple but I dont know the answer. > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > acetate the effect is same. > I dont know why this Zn in not compatible with HEPES. > Could you please tell me why is this? > I appreciate your help. > > Thanks > Rajesh > -- * * * James Kiefer, Ph.D. * Structural Biology Genentech, Inc. 1 DNA Way, Mailstop 27 South San Francisco, CA 94080-4990
Re: [ccp4bb] zinc with HEPES
You are correct. My Friday frazzled brain is stuck in pharmacology mode so I was thinking not of free hydroxide concentration but in terms of potential exchangable groups being sequestered by the buffer. No more posting on Friday. My apologies. Katherine On Fri, May 11, 2012 at 12:23 PM, Jacob Keller < j-kell...@fsm.northwestern.edu> wrote: > Just to make sure I understand pH correctly: isn't it true that the [OH-] > should always be the same at a given pH (by definition)? > > JPK > > On Fri, May 11, 2012 at 11:48 AM, Katherine Sippel < > katherine.sip...@gmail.com> wrote: > >> That is probably because you pH Tris with HCl rather than HEPES with >> NaOH. The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are probably >> what are killing your solution. >> >> Cheers, >> >> Katherine >> >> >> On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar wrote: >> >>> If there is no cure , then fine. >>> pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6. >>> Thanks to every one >>> Rajesh >>> >>> >>> -- >>> Date: Fri, 11 May 2012 12:35:45 -0400 >>> From: dj...@cornell.edu >>> >>> Subject: Re: [ccp4bb] zinc with HEPES >>> To: CCP4BB@JISCMAIL.AC.UK >>> >>> >>> >>> There is no cure for HEPES. >>> >>> >>> >>> >>> -- >>> === >>> All Things Serve the Beam >>> === >>>David J. Schuller >>>modern man in a post-modern world >>>MacCHESS, Cornell University >>>schul...@cornell.edu >>> >>> >> > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > *** >
Re: [ccp4bb] zinc with HEPES
Jacob Keller wrote:
Just to make sure I understand pH correctly: isn't it true that the
[OH-] should always be the same at a given pH (by definition)?
Maybe not by definition but by equilibration with [H+] to make water:
[H][OH]/[H2O] = Kw
[OH] = Kw[H2O]/[H]
if [W] is taken as unity then Kw is ~ 10^-14
[OH] = (~10^-14)/[H]
pOH = ~14 - pH
If neutrality is when [H] = [OH] that would be when pH = pKw/2
which is _approximately_ 7 depending on temperature, ionic strength etc.
(some students get the idea that neutrality is defined to be pH == 7.)
JPK
On Fri, May 11, 2012 at 11:48 AM, Katherine Sippel
mailto:katherine.sip...@gmail.com>> wrote:
That is probably because you pH Tris with HCl rather than HEPES with
NaOH. The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are
probably what are killing your solution.
Cheers,
Katherine
On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar mailto:ccp4...@hotmail.com>> wrote:
If there is no cure , then fine.
pH may not be the answer as it doesn't Happen with TRIS buffer
pH 7.6.
Thanks to every one
Rajesh
Date: Fri, 11 May 2012 12:35:45 -0400
From: dj...@cornell.edu <mailto:dj...@cornell.edu>
Subject: Re: [ccp4bb] zinc with HEPES
To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
There is no cure for HEPES.
--
===
All Things Serve the Beam
===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu
<mailto:schul...@cornell.edu>
--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu <mailto:j-kell...@northwestern.edu>
***
Re: [ccp4bb] zinc with HEPES/seeding
It sounds as though microseeding worked very well, but the crystals are still growing far too quickly. Try diluting the protein and/or the reservoir solution to half the concs you are using or lower. To have enough protein you need larger drops, say 500 + 500 + 200 by hand if you can't do it with the Phoenix (wrong robot of course ;) Dispense the seed with a Hamilton syringe, rinsing the needle in the reservoirs before adding to the drops. You could also try using large volumes, say 4 ul total, with dilute ingredients, and make a small hole in the tape with a pin to let the wells slowly dry out. On 11 May 2012 18:35, Rajesh Kumar wrote: > > Dear Patrick, > > You along with others had made some suggestions last time. May be its a > good time to update. > > With classical screening, I got a crystal like appearances/shower with > HEPES 7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and > with different conc of Lithium I could only get very very thin needles > which shower and difficult to pick even with 0.05 loop. changing conc of > protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs, > glycerol, ethylene glycol, didnt reduce shower. > > I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS > screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) > 100nl+100nL+50nL seeds using Phoenix. > > Got several nice hits which very good size individual crystals in > conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. > When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but > no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after > plates were set and there was no luck. Crystals started to appear from 20 > min onwards and keep growing in next couple of hours. I thought of trying > dehydration but they were already in dehydrating conditions them selves. I > wanted to ask if anyone ever failed with MMS but thought not > waste others time on this. Cross seeding to full length protein and Se met > protein also gave beautiful crystal but again no diffraction. I have not > checked SeMet as they were bit small. > > I am still open to ideas if you have any thing on seeding. I did try in > microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul > seeds, same as sitting drop which gave me very good looking crystals) but I > didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but > it didn't give me anything. > > Currently, I am making entropy mutations, new constructs > of different lengths to solve the above problem. > > I still want to improve this condition with needles, because I collected a > 4.5A data on one of the needle (just one). I need phases. I have sent some > Iodide soaks to synchotron (yet to collect data) but manipulating these > crystal drops with more than 100 tiny needles with a tough membrane on it > has been frustrating as I end up loosing several drops to just to fish out > 1-2 needles. I am ready to try if there any trick left. > > Thanks for lots and lots of help. > > Regards, > Rajesh > -- > Date: Fri, 11 May 2012 18:09:54 +0100 > Subject: Re: [ccp4bb] zinc with HEPES > From: patr...@douglas.co.uk > To: ccp4...@hotmail.com > > Rajesh > > How did you do the MMS? By hand or with a robot, and what screens did you > use? > > and why did you change to HEPES out of interest? > > Patrick > > > On 11 May 2012 18:05, Rajesh Kumar wrote: > > The rationale was to see if Zn could make differences in crystal > morphology. This is because the protein has CxxC and CxxH similar to a zinc > finger motif. > All my efforts, additive screening, MMS, streaking, micro batch, hanging > drop, changing drop ratio, drop shape, did not help me to either increase > thickness or change the shape of very very thin needle crystals. > Yes, I will try very less, 50uM. > Thanks for helping me to understand. > Rajesh > > > Date: Fri, 11 May 2012 12:53:53 -0400 > > Subject: Re: [ccp4bb] zinc with HEPES > > From: liehy...@gmail.com > > To: ccp4...@hotmail.com > > > > Rajesh, > > 10mM zinc seems a bit too high. I normally used it at <50uM conc. > > ray > > > > On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar > wrote: > > > Dear All, > > > > > > This question sounds simple but I dont know the answer. > > > I was preparing a 24 well crystal screen. When I try to use 10 mM > ZnSO4 > > > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > > > acetate the effect is same. > > > I dont know why this Zn in not compatible with HEPES. > > > Co
Re: [ccp4bb] zinc with HEPES/seeding
Thanks. I will try. Rajesh Date: Fri, 11 May 2012 12:38:20 -0500 From: j-kell...@fsm.northwestern.edu Subject: Re: [ccp4bb] zinc with HEPES/seeding To: CCP4BB@JISCMAIL.AC.UK mitegen loops might help, particularly micromesh... JPK On Fri, May 11, 2012 at 12:35 PM, Rajesh Kumar wrote: Dear Patrick, You along with others had made some suggestions last time. May be its a good time to update. With classical screening, I got a crystal like appearances/shower with HEPES 7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and with different conc of Lithium I could only get very very thin needles which shower and difficult to pick even with 0.05 loop. changing conc of protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs, glycerol, ethylene glycol, didnt reduce shower. I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) 100nl+100nL+50nL seeds using Phoenix. Got several nice hits which very good size individual crystals in conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after plates were set and there was no luck. Crystals started to appear from 20 min onwards and keep growing in next couple of hours. I thought of trying dehydration but they were already in dehydrating conditions them selves. I wanted to ask if anyone ever failed with MMS but thought not waste others time on this. Cross seeding to full length protein and Se met protein also gave beautiful crystal but again no diffraction. I have not checked SeMet as they were bit small. I am still open to ideas if you have any thing on seeding. I did try in microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul seeds, same as sitting drop which gave me very good looking crystals) but I didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but it didn't give me anything. Currently, I am making entropy mutations, new constructs of different lengths to solve the above problem. I still want to improve this condition with needles, because I collected a 4.5A data on one of the needle (just one). I need phases. I have sent some Iodide soaks to synchotron (yet to collect data) but manipulating these crystal drops with more than 100 tiny needles with a tough membrane on it has been frustrating as I end up loosing several drops to just to fish out 1-2 needles. I am ready to try if there any trick left. Thanks for lots and lots of help. Regards,Rajesh Date: Fri, 11 May 2012 18:09:54 +0100 Subject: Re: [ccp4bb] zinc with HEPES From: patr...@douglas.co.uk To: ccp4...@hotmail.com Rajesh How did you do the MMS? By hand or with a robot, and what screens did you use? and why did you change to HEPES out of interest? Patrick On 11 May 2012 18:05, Rajesh Kumar wrote: The rationale was to see if Zn could make differences in crystal morphology. This is because the protein has CxxC and CxxH similar to a zinc finger motif.All my efforts, additive screening, MMS, streaking, micro batch, hanging drop, changing drop ratio, drop shape, did not help me to either increase thickness or change the shape of very very thin needle crystals. Yes, I will try very less, 50uM.Thanks for helping me to understand.Rajesh > Date: Fri, 11 May 2012 12:53:53 -0400 > Subject: Re: [ccp4bb] zinc with HEPES > From: liehy...@gmail.com > To: ccp4...@hotmail.com > > Rajesh, > 10mM zinc seems a bit too high. I normally used it at <50uM conc. > ray > > On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar wrote: > > Dear All, > > > > This question sounds simple but I dont know the answer. > > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 > > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > > acetate the effect is same. > > I dont know why this Zn in not compatible with HEPES. > > Could you please tell me why is this? > > I appreciate your help. > > > > Thanks > > Rajesh -- patr...@douglas.co.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] zinc with HEPES/seeding
mitegen loops might help, particularly micromesh... JPK On Fri, May 11, 2012 at 12:35 PM, Rajesh Kumar wrote: > > Dear Patrick, > > You along with others had made some suggestions last time. May be its a > good time to update. > > With classical screening, I got a crystal like appearances/shower with > HEPES 7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and > with different conc of Lithium I could only get very very thin needles > which shower and difficult to pick even with 0.05 loop. changing conc of > protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs, > glycerol, ethylene glycol, didnt reduce shower. > > I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS > screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) > 100nl+100nL+50nL seeds using Phoenix. > > Got several nice hits which very good size individual crystals in > conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. > When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but > no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after > plates were set and there was no luck. Crystals started to appear from 20 > min onwards and keep growing in next couple of hours. I thought of trying > dehydration but they were already in dehydrating conditions them selves. I > wanted to ask if anyone ever failed with MMS but thought not > waste others time on this. Cross seeding to full length protein and Se met > protein also gave beautiful crystal but again no diffraction. I have not > checked SeMet as they were bit small. > > I am still open to ideas if you have any thing on seeding. I did try in > microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul > seeds, same as sitting drop which gave me very good looking crystals) but I > didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but > it didn't give me anything. > > Currently, I am making entropy mutations, new constructs > of different lengths to solve the above problem. > > I still want to improve this condition with needles, because I collected a > 4.5A data on one of the needle (just one). I need phases. I have sent some > Iodide soaks to synchotron (yet to collect data) but manipulating these > crystal drops with more than 100 tiny needles with a tough membrane on it > has been frustrating as I end up loosing several drops to just to fish out > 1-2 needles. I am ready to try if there any trick left. > > Thanks for lots and lots of help. > > Regards, > Rajesh > -- > Date: Fri, 11 May 2012 18:09:54 +0100 > Subject: Re: [ccp4bb] zinc with HEPES > From: patr...@douglas.co.uk > To: ccp4...@hotmail.com > > Rajesh > > How did you do the MMS? By hand or with a robot, and what screens did you > use? > > and why did you change to HEPES out of interest? > > Patrick > > > On 11 May 2012 18:05, Rajesh Kumar wrote: > > The rationale was to see if Zn could make differences in crystal > morphology. This is because the protein has CxxC and CxxH similar to a zinc > finger motif. > All my efforts, additive screening, MMS, streaking, micro batch, hanging > drop, changing drop ratio, drop shape, did not help me to either increase > thickness or change the shape of very very thin needle crystals. > Yes, I will try very less, 50uM. > Thanks for helping me to understand. > Rajesh > > > Date: Fri, 11 May 2012 12:53:53 -0400 > > Subject: Re: [ccp4bb] zinc with HEPES > > From: liehy...@gmail.com > > To: ccp4...@hotmail.com > > > > Rajesh, > > 10mM zinc seems a bit too high. I normally used it at <50uM conc. > > ray > > > > On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar > wrote: > > > Dear All, > > > > > > This question sounds simple but I dont know the answer. > > > I was preparing a 24 well crystal screen. When I try to use 10 mM > ZnSO4 > > > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > > > acetate the effect is same. > > > I dont know why this Zn in not compatible with HEPES. > > > Could you please tell me why is this? > > > I appreciate your help. > > > > > > Thanks > > > Rajesh > > > > > -- > patr...@douglas.co.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] zinc with HEPES/seeding
Dear Patrick, You along with others had made some suggestions last time. May be its a good time to update. With classical screening, I got a crystal like appearances/shower with HEPES 7.5 and LiSo4 1.5M. Trying to vary the pH of Hepes or using Tris and with different conc of Lithium I could only get very very thin needles which shower and difficult to pick even with 0.05 loop. changing conc of protein, salt, adding oil on well, changing drop ratio, adding 5% PEGs, glycerol, ethylene glycol, didnt reduce shower. I prepared the seeds from 7.6 ph and 1.25 M Liso4 conditions and MMS screened with all the 4 screens (Qiagen procomplex, classic, peg, JCSG) 100nl+100nL+50nL seeds using Phoenix. Got several nice hits which very good size individual crystals in conditions with 30% glycerol and 14% Isopropanol, 20% PEG8K, 30 PEG400. When I optimized them I got beautiful crystals and tried at ALS 5.0.3 but no spots. I tried picking crystals from 30 min to 4 hrs to 4 days after plates were set and there was no luck. Crystals started to appear from 20 min onwards and keep growing in next couple of hours. I thought of trying dehydration but they were already in dehydrating conditions them selves. I wanted to ask if anyone ever failed with MMS but thought not waste others time on this. Cross seeding to full length protein and Se met protein also gave beautiful crystal but again no diffraction. I have not checked SeMet as they were bit small. I am still open to ideas if you have any thing on seeding. I did try in microbatch and hanging drop as well (1.5 ul protein+ 1.4ul reservor+ 0.4 ul seeds, same as sitting drop which gave me very good looking crystals) but I didn't get any thing. I tried streaking with reduced LiSO4 to 1.1 M but it didn't give me anything. Currently, I am making entropy mutations, new constructs of different lengths to solve the above problem. I still want to improve this condition with needles, because I collected a 4.5A data on one of the needle (just one). I need phases. I have sent some Iodide soaks to synchotron (yet to collect data) but manipulating these crystal drops with more than 100 tiny needles with a tough membrane on it has been frustrating as I end up loosing several drops to just to fish out 1-2 needles. I am ready to try if there any trick left. Thanks for lots and lots of help. Regards,Rajesh Date: Fri, 11 May 2012 18:09:54 +0100 Subject: Re: [ccp4bb] zinc with HEPES From: patr...@douglas.co.uk To: ccp4...@hotmail.com Rajesh How did you do the MMS? By hand or with a robot, and what screens did you use? and why did you change to HEPES out of interest? Patrick On 11 May 2012 18:05, Rajesh Kumar wrote: The rationale was to see if Zn could make differences in crystal morphology. This is because the protein has CxxC and CxxH similar to a zinc finger motif.All my efforts, additive screening, MMS, streaking, micro batch, hanging drop, changing drop ratio, drop shape, did not help me to either increase thickness or change the shape of very very thin needle crystals. Yes, I will try very less, 50uM.Thanks for helping me to understand.Rajesh > Date: Fri, 11 May 2012 12:53:53 -0400 > Subject: Re: [ccp4bb] zinc with HEPES > From: liehy...@gmail.com > To: ccp4...@hotmail.com > > Rajesh, > 10mM zinc seems a bit too high. I normally used it at <50uM conc. > ray > > On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar wrote: > > Dear All, > > > > This question sounds simple but I dont know the answer. > > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 > > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > > acetate the effect is same. > > I dont know why this Zn in not compatible with HEPES. > > Could you please tell me why is this? > > I appreciate your help. > > > > Thanks > > Rajesh -- patr...@douglas.co.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] zinc with HEPES
Almost assuredly you are getting zinc hydroxide precipitate. Zn(OH)2 will readily precipitate from solutions of alkaline Zn(II). Your problem is compounded by the fact that HEPES is a non-coordinating buffer, so it does not help solubilize the zinc ion. You might find a weakly coordinating buffer, like Tris, will help solubilize Zn(II) at a sufficiently low concentration. Or you could lower the pH slightly to lower the hydroxide ion concentration and increase the solubility of Zn(II). You could also add a chelator like EDTA, tartrate, etc. to the solution to solubilize Zn(II) under alkaline conditions, but then you have the problem of hoping that your protein has a higher affinity for the metal ion than the chelator. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 5/11/2012 12:26 PM, Rajesh Kumar wrote: Dear All, This question sounds simple but I dont know the answer. I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn acetate the effect is same. I dont know why this Zn in not compatible with HEPES. Could you please tell me why is this? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] zinc with HEPES
Just to make sure I understand pH correctly: isn't it true that the [OH-] should always be the same at a given pH (by definition)? JPK On Fri, May 11, 2012 at 11:48 AM, Katherine Sippel < katherine.sip...@gmail.com> wrote: > That is probably because you pH Tris with HCl rather than HEPES with NaOH. > The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are probably what > are killing your solution. > > Cheers, > > Katherine > > > On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar wrote: > >> If there is no cure , then fine. >> pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6. >> Thanks to every one >> Rajesh >> >> >> -- >> Date: Fri, 11 May 2012 12:35:45 -0400 >> From: dj...@cornell.edu >> >> Subject: Re: [ccp4bb] zinc with HEPES >> To: CCP4BB@JISCMAIL.AC.UK >> >> >> >> There is no cure for HEPES. >> >> >> >> >> -- >> === >> All Things Serve the Beam >> === >>David J. Schuller >>modern man in a post-modern world >>MacCHESS, Cornell University >>schul...@cornell.edu >> >> > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] zinc with HEPES
The rationale was to see if Zn could make differences in crystal morphology. This is because the protein has CxxC and CxxH similar to a zinc finger motif.All my efforts, additive screening, MMS, streaking, micro batch, hanging drop, changing drop ratio, drop shape, did not help me to either increase thickness or change the shape of very very thin needle crystals. Yes, I will try very less, 50uM.Thanks for helping me to understand.Rajesh > Date: Fri, 11 May 2012 12:53:53 -0400 > Subject: Re: [ccp4bb] zinc with HEPES > From: liehy...@gmail.com > To: ccp4...@hotmail.com > > Rajesh, > 10mM zinc seems a bit too high. I normally used it at <50uM conc. > ray > > On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar wrote: > > Dear All, > > > > This question sounds simple but I dont know the answer. > > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 > > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > > acetate the effect is same. > > I dont know why this Zn in not compatible with HEPES. > > Could you please tell me why is this? > > I appreciate your help. > > > > Thanks > > Rajesh
Re: [ccp4bb] zinc with HEPES
That is probably because you pH Tris with HCl rather than HEPES with NaOH. The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are probably what are killing your solution. Cheers, Katherine On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar wrote: > If there is no cure , then fine. > pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6. > Thanks to every one > Rajesh > > > -- > Date: Fri, 11 May 2012 12:35:45 -0400 > From: dj...@cornell.edu > > Subject: Re: [ccp4bb] zinc with HEPES > To: CCP4BB@JISCMAIL.AC.UK > > > > There is no cure for HEPES. > > > > > -- > === > All Things Serve the Beam > === >David J. Schuller >modern man in a post-modern world >MacCHESS, Cornell University >schul...@cornell.edu > >
Re: [ccp4bb] zinc with HEPES
pH is the culprit here Like some already mentioned change your pH to 6.4 and use a different buffer like cacodylate or you can use Zinc acetate in water pH still would be 6.4 from your last mail why would you add Zn to your initial hit condition is there any rationale? Padayatti On Fri, May 11, 2012 at 12:26 PM, Rajesh Kumar wrote: > Dear All, > > This question sounds simple but I dont know the answer. > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > acetate the effect is same. > I dont know why this Zn in not compatible with HEPES. > Could you please tell me why is this? > I appreciate your help. > > Thanks > Rajesh -- Pius S Padayatti,PhD, Phone: 216-658-4528
Re: [ccp4bb] zinc with HEPES
If there is no cure , then fine.pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6.Thanks to every oneRajesh Date: Fri, 11 May 2012 12:35:45 -0400 From: dj...@cornell.edu Subject: Re: [ccp4bb] zinc with HEPES To: CCP4BB@JISCMAIL.AC.UK There is no cure for HEPES. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] zinc with HEPES
Initial screen was 0.1 M Hepes 7.5 and 1.25 M LiSO4. I added ZnSO4 to HEPES and immediately it precipitated.Just now I tried to add 10 mM ZnSO4 in to tube after volume is made up and only with HEPES 100mM pH 7.5.It happens as earlier. So is there any way I could use Zn with HEPES. I thought many might have used this combination. ThanksRajesh Date: Fri, 11 May 2012 11:29:03 -0500 Subject: Re: [ccp4bb] zinc with HEPES From: j-kell...@fsm.northwestern.edu To: ccp4...@hotmail.com CC: CCP4BB@jiscmail.ac.uk Try adding water first, so that you are not mixing concentrated Zn with concentrated HEPES. Also it depends what else is in your cocktail. JPK On Fri, May 11, 2012 at 11:26 AM, Rajesh Kumar wrote: Dear All, This question sounds simple but I dont know the answer.I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn acetate the effect is same. I dont know why this Zn in not compatible with HEPES.Could you please tell me why is this?I appreciate your help. Thanks Rajesh -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] zinc with HEPES
On 05/11/12 12:26, Rajesh Kumar wrote: Dear All, This question sounds simple but I dont know the answer. I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn acetate the effect is same. I dont know why this Zn in not compatible with HEPES. Could you please tell me why is this? I appreciate your help. There is no cure for HEPES. -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] zinc with HEPES
Could it be that the pH is too high? Zn is amphoteric and you might be seeing a precipitate of Zn(OH)2. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Rajesh Kumar [ccp4...@hotmail.com] Sent: Friday, May 11, 2012 7:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] zinc with HEPES Dear All, This question sounds simple but I dont know the answer. I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn acetate the effect is same. I dont know why this Zn in not compatible with HEPES. Could you please tell me why is this? I appreciate your help. Thanks Rajesh
Re: [ccp4bb] zinc with HEPES
Try adding water first, so that you are not mixing concentrated Zn with concentrated HEPES. Also it depends what else is in your cocktail. JPK On Fri, May 11, 2012 at 11:26 AM, Rajesh Kumar wrote: > Dear All, > > This question sounds simple but I dont know the answer. > I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 > with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn > acetate the effect is same. > I dont know why this Zn in not compatible with HEPES. > Could you please tell me why is this? > I appreciate your help. > > Thanks > Rajesh > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] zinc with HEPES
Dear All, This question sounds simple but I dont know the answer.I was preparing a 24 well crystal screen. When I try to use 10 mM ZnSO4 with HEPES (pH 7.6) buffer it precipitates. I tried both ZnCl2 and Zn acetate the effect is same. I dont know why this Zn in not compatible with HEPES.Could you please tell me why is this?I appreciate your help. ThanksRajesh
