Re: [ccp4bb] Desalting columns
Actually, I would refer the ccp4-bbs to Journal of Structural Biology 175 (2011) pp216-223 for the use of fluorescence in relation to protein crystallization. Regards, Bryan -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Christian Roth Sent: Tuesday, February 28, 2012 8:09 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Desalting columns If you want test a lot of different conditions a Thermofluorecence assay might work for your protein and you may find a condition which stabilise your protein. However there is no warranty that it crystallise better afterwards. Christian Am Montag 27 Februar 2012 17:01:33 schrieb Sangeetha Vedula: > Dear bb users, > > I am trying to crystallize a ~320 kDa protein that crashes out if > concentrated past about 3 mg/mL. > > I would like to try to exchange it into various buffer-salt-additive > combinations to see which buffer works. For a starting point, I'd like to > use desalting colums. > > Does anyone have suggestions for good buffer exchange and sample recovery? > I woud like to load about 250 uL onto each column. > > Thanks a lot! > > Best regards, > > Sangeetha. >
Re: [ccp4bb] Desalting columns
If you want test a lot of different conditions a Thermofluorecence assay might work for your protein and you may find a condition which stabilise your protein. However there is no warranty that it crystallise better afterwards. Christian Am Montag 27 Februar 2012 17:01:33 schrieb Sangeetha Vedula: > Dear bb users, > > I am trying to crystallize a ~320 kDa protein that crashes out if > concentrated past about 3 mg/mL. > > I would like to try to exchange it into various buffer-salt-additive > combinations to see which buffer works. For a starting point, I'd like to > use desalting colums. > > Does anyone have suggestions for good buffer exchange and sample recovery? > I woud like to load about 250 uL onto each column. > > Thanks a lot! > > Best regards, > > Sangeetha. >
Re: [ccp4bb] Desalting columns
Ø in 2004. Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml. That is not necessarily the success rate for low concentrations, which we actually would like to have. We would need negatives for < 3 for to give a correct answer. I guess even occurrence might be more frequent now. Something for our center data miners who have the negatives kept . Anecdotally, my personal below-3-occurance not success, that is 100% is ~15%, but that does not mean much. Even if it were the success rate: 5% chance compared to zero chance when precipitated Id take it and probably learn something useful during the experiment. BR
Re: [ccp4bb] Desalting columns
Hi, We have a case where a 260 KDa protein crystallized at 3 mg/ml, that was the highest concentration we could achieve, without playing with the protein buffer. Protein-to-well-solution ratio was 3:1. When screening, we tested all ratios between 4:1 and 1:1. Crystals only appeared at 3:1 ratio. That is another important variable to test, I guess. Regards, -Andre. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp (Hofkristallrat a.D.) [hofkristall...@gmail.com] Sent: Monday, February 27, 2012 1:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Desalting columns Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? For crystallization, the concentration needs to be a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. There is ample evidence for proteins crystallizing below 3 mg/ml. The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. Sometimes the shape of a distribution matters ;-) BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sangeetha Vedula Sent: Monday, February 27, 2012 8:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Desalting columns Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
We did some data mining from remark 280 of the PDB in 2004. Of the 1000 entries that listed [protein], 46 proteins were crystallized below 3.1 mg/ml. See table 3 at http://www.douglas.co.uk/PDB_data.htm . Patrick On 27 February 2012 16:25, Bernhard Rupp (Hofkristallrat a.D.) < hofkristall...@gmail.com> wrote: > Why, in the first place, do you feel an urge to concentrate your protein > above 3 mg/ml ? > > ** ** > > For crystallization, the concentration needs to be > > **a) **high enough to achieve supersaturation, meaning close enough > to the maximum solubility in a given buffer so that the precipitant can > drive the system in to supersaturation, preferably of a level where > homogenous nucleation can occur (or you micro-seed, if necessary) > > **b) **high enough that sufficient material for crystals of > acceptable size to grow is in the drop, which is generally the case, lest > micro-crystal showers happen. > > ** ** > > There is ample evidence for proteins crystallizing below 3 mg/ml. > > ** ** > > The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased > towards highly soluble, smaller (lower hanging fruit) proteins. > > ** ** > > Sometimes the shape of a distribution matters ;-) > > ** ** > > BR > > ** ** > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Sangeetha > Vedula > *Sent:* Monday, February 27, 2012 8:02 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *Subject:* [ccp4bb] Desalting columns > > ** ** > > Dear bb users, > > I am trying to crystallize a ~320 kDa protein that crashes out if > concentrated past about 3 mg/mL. > > I would like to try to exchange it into various buffer-salt-additive > combinations to see which buffer works. For a starting point, I'd like to > use desalting colums. > > Does anyone have suggestions for good buffer exchange and sample recovery? > I woud like to load about 250 uL onto each column. > > Thanks a lot! > > Best regards, > > Sangeetha. > -- 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] Desalting columns
Hi Sangeetha: If you just want to check which buffer is good for your protein, maybe you can try to set up a crystallization screen, keeping your protein concentration just 3 mg/ml. You can observe (after several days) which conditions give you a clear drop, and maybe you can find a clue which buffer is better for your protein. If you want to speed up the whole processes, you can also add glycerol into the drops to grasp the water molecules. Yu Xiaodi Date: Mon, 27 Feb 2012 11:01:33 -0500 From: sangeetha...@gmail.com Subject: [ccp4bb] Desalting columns To: CCP4BB@JISCMAIL.AC.UK Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
Why, in the first place, do you feel an urge to concentrate your protein above 3 mg/ml ? For crystallization, the concentration needs to be a) high enough to achieve supersaturation, meaning close enough to the maximum solubility in a given buffer so that the precipitant can drive the system in to supersaturation, preferably of a level where homogenous nucleation can occur (or you micro-seed, if necessary) b) high enough that sufficient material for crystals of acceptable size to grow is in the drop, which is generally the case, lest micro-crystal showers happen. There is ample evidence for proteins crystallizing below 3 mg/ml. The often quoted PDB/BMCD average of somewhere around 10 mg/ml is biased towards highly soluble, smaller (lower hanging fruit) proteins. Sometimes the shape of a distribution matters ;-) BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sangeetha Vedula Sent: Monday, February 27, 2012 8:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Desalting columns Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. ~ 1 ml spin columns with Sephadex G25 or Biogel P6. Typically, when loading 200 ul of the sample onto 1 ml column, desalting efficiency is ~ 95% and protein recovery is ~ 90-95% without volume change. Lowering sample volume will increasing dealting and reduce yield but I never had the yeild worse than 75% even with 50 ul volume. Bio-Rad sells prepacked "Bio-Spin P-6" or you can buy empty columns and dry P6 (by "fine" grade in this case) and pack to 1.2 ml per column, which will take your 250 ul volume and still result in decent desalting. Pharmacia probably sells something similar and many companies sell 0.5 ml spin columns. I like P6 (polyacrylamide) better than Sephadex (dextran) because usually (but not always) non-specific binding is lower. - Dima Thanks a lot! Best regards, Sangeetha.
Re: [ccp4bb] Desalting columns
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Sangeetha, provided you express the protein in E. coli, you could also sonicate the cell debris in various buffers and compare supernatant/pellet on SDS-gel. It might already give you a first clue and is faster, cheaper and you don't risk to clog the new columns with aggregated protein. Tim On 02/27/2012 05:01 PM, Sangeetha Vedula wrote: > Dear bb users, > > I am trying to crystallize a ~320 kDa protein that crashes out if > concentrated past about 3 mg/mL. > > I would like to try to exchange it into various buffer-salt-additive > combinations to see which buffer works. For a starting point, I'd like to > use desalting colums. > > Does anyone have suggestions for good buffer exchange and sample recovery? > I woud like to load about 250 uL onto each column. > > Thanks a lot! > > Best regards, > > Sangeetha. > - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFPS6xEUxlJ7aRr7hoRAqOqAJ0cYn4488ctcIRUeZa4K3f2smojfQCeKBDI +nO8deQmkPNB9nWSWJtBoOI= =SU1z -END PGP SIGNATURE-
[ccp4bb] Desalting columns
Dear bb users, I am trying to crystallize a ~320 kDa protein that crashes out if concentrated past about 3 mg/mL. I would like to try to exchange it into various buffer-salt-additive combinations to see which buffer works. For a starting point, I'd like to use desalting colums. Does anyone have suggestions for good buffer exchange and sample recovery? I woud like to load about 250 uL onto each column. Thanks a lot! Best regards, Sangeetha.
