Re: [ccp4bb] Suggestions for Reducing Protein Precipitation
Hi Matt, if you have a crystallisation robot (and if you haven't you can still do this manually) you can use a simple microbatch under oil experiment to determine the likely optimum pH and salt concentration. Just set up a grid across pH (I usually use a phosphate buffer for this as it is easiest to obtain a pH range with few solutions, and then switch back to a more suitable buffer once I know the ideal pH), with varying salt concentrations (usually between 0 and 1000mM in 100mM steps) for each pH point. Set your drops up as you would any microbatch experiment with 50:50 ratio of your protein to the condition and a drop size of 1 or 2 ul. After set up you simply inspect the drops under the microscope, usually immediately and then again after a few hours (or overnight) at your intended working temperature(s). Record where you see clear drops and where you see signs of precipitation, and if you plot them you should end up with a curve showing you the optimum pH and salt. You can generally use your protein at fairly low concentration for this (you often have too because your buffer is suboptimal) so it is quite economical. In addition you can repeat it with different salts or additives as desired, and gradually refine the condition as necessary, and of course you could always combine this general approach with some of the other screens suggested. For a more detailed explanation / example check out: http://www.ncbi.nlm.nih.gov/pubmed/12832769 Acta Cryst. (2003). D59, 1230-1233 Expression, purification and preliminary X-ray analysis of the BRCT domain from Rhp9/Crb2 J. A. Hinks, M. Roe, J. C. Y. Ho, F. Z. Watts, J. Phelan, M. McAllister and L. H. Pearl Hope that's useful, John. john.hi...@syngenta.com www.syngenta.com -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of David Briggs Sent: 12 August 2010 20:22 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Suggestions for Reducing Protein Precipitation Hi Matt, Have you tried changing the pH? Is it possible that at pH 8 you are at the pI of your protein (i.e. where it has zero net charge and is at its least soluble) ? I have also read a paper (can't find the reference right now) where the authors precipitated their protein by dialysing into water, and then aliquoted the suspended precipitate (suitable amounts to allow concentration measurement by OD280 - if you've got a nanodrop or similar, you only need a few ul). The precipitate was pelleted by centrifugation, and the water was removed. A series of different buffer types / salt types / strengths were added to each aliquot to resolubilise the protein. The tubes were briefly vortexed and left at room temp for 30mins(?) any remaining precipitate was pelleted again, and the OD280 of the supernatant measured. The buffer with the highest OD in solution allowed them to reach higher concentrations of protein for crystal screening. Hope this makes sense - contact me if you need more info, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 12 August 2010 19:57, Matthew Bratkowski mab...@cornell.edu wrote: Hi. I am working with a protein that has difficulties staying in solution when concentrated. The buffers that I have been using contain 20 mM Tris pH 8.0, 5 mM BME (or 2 mM DTT), 10% Glycerol, and NaCl from 50 mM up to 2 M. The protein seems fine to dialyze into low salt buffer (50 mM NaCl) when dilute, but seems to precipitate onto an anion exchange column during loading, as noticed by brown spots on the column and a reduction in yield after the run. Despite these issues, I have been able to concentrate the protein to about 18 mg/mL after removing glycerol and using 200 mM NaCl and 2 mM DTT. However, after sitting at 4 C for a few hours, I again notice precipitate in the concentrated stock. I figure that I will probably just work with more diluted protein next time, but was concerned that my protein was not stable enough in this buffer to use for crystallization. I am seeking advice on finding a better buffer for the protein. Can anyone suggest whether making subtle buffer changes, such as using HEPES instead of Tris or KCl instead of NaCl would be advantageous? Additionally, I was interested I find out what concentration of glycerol or NaCl could still be added for the protein to still be usable for crystallization, as well as any additives that will prevent precipitation but not interfere with crystallization. Also, could someone suggest a good way to test protein stability in various buffers on a small scale before deciding
Re: [ccp4bb] Suggestions for Reducing Protein Precipitation
Hi Matt, Check out the following paper and some screens available commercially based on these: Acta Crystallogr D Biol Crystallogr. 2004 Sep;60(Pt 9):1670-3. Epub 2004 Aug 26. Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Jancarik J, Pufan R, Hong C, Kim SH, Kim R. Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005 Dec 1;61(Pt 12):1035-8. Epub 2005 Nov 5. Crystallization Optimum Solubility Screening: using crystallization results to identify the optimal buffer for protein crystal formation. Collins B, Stevens RC, Page R. The first one is available as a kit (JBS solubility screen): http://www.jenabioscience.com/cms/en/1/catalog/739//Search http://www.mitegen.com/mic_catalog.php?c=jenCrystaloptSolukit It appears that you have not yet set up screens on this protein. I think it is worth trying: a) Setting up crystallization screens under dilute conditions. Some proteins crystallize under such conditions. b) Set up screens at the higher concentration immediately after concentrating before precipitation starts occurring. c) Try adding some detergent and I think glycerol should help to improve solubility You can also check out Hampton's PCT Pre-Crystallization Test. Thanks, -Debanu. From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Matthew Bratkowski Sent: Thursday, August 12, 2010 11:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Suggestions for Reducing Protein Precipitation Hi. I am working with a protein that has difficulties staying in solution when concentrated. The buffers that I have been using contain 20 mM Tris pH 8.0, 5 mM BME (or 2 mM DTT), 10% Glycerol, and NaCl from 50 mM up to 2 M. The protein seems fine to dialyze into low salt buffer (50 mM NaCl) when dilute, but seems to precipitate onto an anion exchange column during loading, as noticed by brown spots on the column and a reduction in yield after the run. Despite these issues, I have been able to concentrate the protein to about 18 mg/mL after removing glycerol and using 200 mM NaCl and 2 mM DTT. However, after sitting at 4 C for a few hours, I again notice precipitate in the concentrated stock. I figure that I will probably just work with more diluted protein next time, but was concerned that my protein was not stable enough in this buffer to use for crystallization. I am seeking advice on finding a better buffer for the protein. Can anyone suggest whether making subtle buffer changes, such as using HEPES instead of Tris or KCl instead of NaCl would be advantageous? Additionally, I was interested I find out what concentration of glycerol or NaCl could still be added for the protein to still be usable for crystallization, as well as any additives that will prevent precipitation but not interfere with crystallization. Also, could someone suggest a good way to test protein stability in various buffers on a small scale before deciding on conditions for a large batch. Thanks, Matt
Re: [ccp4bb] Suggestions for Reducing Protein Precipitation
Hi Matt, Have you tried changing the pH? Is it possible that at pH 8 you are at the pI of your protein (i.e. where it has zero net charge and is at its least soluble) ? I have also read a paper (can't find the reference right now) where the authors precipitated their protein by dialysing into water, and then aliquoted the suspended precipitate (suitable amounts to allow concentration measurement by OD280 - if you've got a nanodrop or similar, you only need a few ul). The precipitate was pelleted by centrifugation, and the water was removed. A series of different buffer types / salt types / strengths were added to each aliquot to resolubilise the protein. The tubes were briefly vortexed and left at room temp for 30mins(?) any remaining precipitate was pelleted again, and the OD280 of the supernatant measured. The buffer with the highest OD in solution allowed them to reach higher concentrations of protein for crystal screening. Hope this makes sense - contact me if you need more info, Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 12 August 2010 19:57, Matthew Bratkowski mab...@cornell.edu wrote: Hi. I am working with a protein that has difficulties staying in solution when concentrated. The buffers that I have been using contain 20 mM Tris pH 8.0, 5 mM BME (or 2 mM DTT), 10% Glycerol, and NaCl from 50 mM up to 2 M. The protein seems fine to dialyze into low salt buffer (50 mM NaCl) when dilute, but seems to precipitate onto an anion exchange column during loading, as noticed by brown spots on the column and a reduction in yield after the run. Despite these issues, I have been able to concentrate the protein to about 18 mg/mL after removing glycerol and using 200 mM NaCl and 2 mM DTT. However, after sitting at 4 C for a few hours, I again notice precipitate in the concentrated stock. I figure that I will probably just work with more diluted protein next time, but was concerned that my protein was not stable enough in this buffer to use for crystallization. I am seeking advice on finding a better buffer for the protein. Can anyone suggest whether making subtle buffer changes, such as using HEPES instead of Tris or KCl instead of NaCl would be advantageous? Additionally, I was interested I find out what concentration of glycerol or NaCl could still be added for the protein to still be usable for crystallization, as well as any additives that will prevent precipitation but not interfere with crystallization. Also, could someone suggest a good way to test protein stability in various buffers on a small scale before deciding on conditions for a large batch. Thanks, Matt
Re: [ccp4bb] Suggestions for Reducing Protein Precipitation
Hi all. I found that reference. :0) Acta Crystallogr D Biol Crystallogr. 2006 Jul;62(Pt 7):833-42. Epub 2006 Jun 20. Assessment of a preliminary solubility screen to improve crystallization trials: uncoupling crystal condition searches. Izaac A, Schall CA, Mueser TC. http://www.ncbi.nlm.nih.gov/pubmed/16790940 They actually used PEG in their test case to force their protein out of solution, but I have used milliQ water successfully in the past. If you are considering using this technique, you probably already know how to precipitate your protein! Good luck! Dave David C. Briggs PhD Father, Structural Biologist and Sceptic University of Manchester E-mail: david.c.bri...@manchester.ac.uk http://manchester.academia.edu/DavidBriggs (v.sensible) http://xtaldave.wordpress.com/ (sensible) http://xtaldave.posterous.com/ (less sensible) Twitter: @xtaldave Skype: DocDCB On 12 August 2010 19:57, Matthew Bratkowski mab...@cornell.edu wrote: Hi. I am working with a protein that has difficulties staying in solution when concentrated. The buffers that I have been using contain 20 mM Tris pH 8.0, 5 mM BME (or 2 mM DTT), 10% Glycerol, and NaCl from 50 mM up to 2 M. The protein seems fine to dialyze into low salt buffer (50 mM NaCl) when dilute, but seems to precipitate onto an anion exchange column during loading, as noticed by brown spots on the column and a reduction in yield after the run. Despite these issues, I have been able to concentrate the protein to about 18 mg/mL after removing glycerol and using 200 mM NaCl and 2 mM DTT. However, after sitting at 4 C for a few hours, I again notice precipitate in the concentrated stock. I figure that I will probably just work with more diluted protein next time, but was concerned that my protein was not stable enough in this buffer to use for crystallization. I am seeking advice on finding a better buffer for the protein. Can anyone suggest whether making subtle buffer changes, such as using HEPES instead of Tris or KCl instead of NaCl would be advantageous? Additionally, I was interested I find out what concentration of glycerol or NaCl could still be added for the protein to still be usable for crystallization, as well as any additives that will prevent precipitation but not interfere with crystallization. Also, could someone suggest a good way to test protein stability in various buffers on a small scale before deciding on conditions for a large batch. Thanks, Matt
