Re: [ccp4bb] Protein concentration for the initial crystallisation trials
Hi, Another way of estimating a starting protein concentration is to watch your concentration process – if your protein is in a spin concentrator (with an appropriate membrane cutoff size say ~ [MW protein]/3) and is losing volume really quickly then keep going. As soon as the concentration starts slowing down try that concentration. Actually, you should really follow the experience of the Oxford SGC where they always set up three drop ratios of protein to reservoir solution – 1:2, 1:1 and 2:1. Cheers, Janet From: CCP4 bulletin board On Behalf Of Edward Snell Sent: Thursday, 9 January 2020 3:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation trials Hi Armando, I echo Rodger’s advice at 10 mg/ml to start. Our high-throughput crystallization lab has a FAQ page that notes this https://hwi.buffalo.edu/high-throughput-crystallization-center/hts-faqs/ but we absolutely recommend looking at conditions where the precipitant concentration varies AND doing a screen with a different protein concentration if sample is available. Both have considerable impact on outcome and the screens we use are designed to provide information on this if they are interpreted correctly. There are a lot of internal references available at https://hwi.buffalo.edu/high-throughput-crystallization-center/crystallization-research/. I would recommend the “What’s in a drop?” paper. If there are any homologous proteins that have been crystallized than those conditions can be a good starting guide. There are some proteins that are far more soluble than typical and can have concentrations almost an order of magnitude greater and the occasional on an order of magnitude less. I don’t remember an absolute study on this but I’m sure there must be as I vaguely remember advice that was size related, smaller proteins requiring higher concentration, larger ones less. This may jog someone’s memory to provide the reference. Forgive me for a blatant advertisement, but there is a very successful crystallization screening service at http://getacrystal.org that screens a large array of conditions in a manner to extract this kind of information from them, provides visual, SONICC and UV imaging, and can study the process at multiple temperatures (very useful from a solubility point of view and preserving samples that may be more transient). We hope to implement MARCO (https://marco.ccr.buffalo.edu/) very soon (which has to be added to the reference list – we slipped up there) so you don’t even need to look at all the images. This is a machine vision system we have been developing with many collaborators – it works! (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198883). As Janet Newman so elegantly said recently, may your New Year Resolutions be high. Best, Eddie Edward Snell Ph.D. Director of the NSF BioXFEL Science and Technology Center President and CEO Hauptman-Woodward Medical Research Institute BioInnovations Chaired Professorship, University at Buffalo, SUNY 700 Ellicott Street, Buffalo, NY 14203-1102 hwi.buffalo.edu Phone: (716) 898 8631 Fax: (716) 898 8660 Skype:eddie.snell Email: esn...@hwi.buffalo.edu<mailto:esn...@hwi.buffalo.edu> Webpage: https://hwi.buffalo.edu/scientist-directory/snell/ [cid:image002.png@01D5C6D4.DD9518C0] Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Wednesday, January 8, 2020 11:29 AM To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation trials I usually set a partial screen, maybe 24-48 conditions. If less than half the wells have precipitate, double protein concentration. If most have precipitate, maybe reduce protein or halve concentration of screen reagents. I usually start at 10 mg/mL or so. You can conveniently change protein conc. by manipulating protein/screen volume ratio. __ Roger Rowlett On Wed, Jan 8, 2020, 11:16 AM Armando Albert mailto:xalb...@iqfr.csic.es>> wrote: Dear all, I was wondering how to guess the optimal protein concentration for the initial crystallisation trials. Is there any trick or assay other than the classic PCT from Hampton? Armando To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the
Re: [ccp4bb] Protein concentration for the initial crystallisation trials
Hi Armando, I echo Rodger’s advice at 10 mg/ml to start. Our high-throughput crystallization lab has a FAQ page that notes this https://hwi.buffalo.edu/high-throughput-crystallization-center/hts-faqs/ but we absolutely recommend looking at conditions where the precipitant concentration varies AND doing a screen with a different protein concentration if sample is available. Both have considerable impact on outcome and the screens we use are designed to provide information on this if they are interpreted correctly. There are a lot of internal references available at https://hwi.buffalo.edu/high-throughput-crystallization-center/crystallization-research/. I would recommend the “What’s in a drop?” paper. If there are any homologous proteins that have been crystallized than those conditions can be a good starting guide. There are some proteins that are far more soluble than typical and can have concentrations almost an order of magnitude greater and the occasional on an order of magnitude less. I don’t remember an absolute study on this but I’m sure there must be as I vaguely remember advice that was size related, smaller proteins requiring higher concentration, larger ones less. This may jog someone’s memory to provide the reference. Forgive me for a blatant advertisement, but there is a very successful crystallization screening service at http://getacrystal.org that screens a large array of conditions in a manner to extract this kind of information from them, provides visual, SONICC and UV imaging, and can study the process at multiple temperatures (very useful from a solubility point of view and preserving samples that may be more transient). We hope to implement MARCO (https://marco.ccr.buffalo.edu/) very soon (which has to be added to the reference list – we slipped up there) so you don’t even need to look at all the images. This is a machine vision system we have been developing with many collaborators – it works! (https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198883). As Janet Newman so elegantly said recently, may your New Year Resolutions be high. Best, Eddie Edward Snell Ph.D. Director of the NSF BioXFEL Science and Technology Center President and CEO Hauptman-Woodward Medical Research Institute BioInnovations Chaired Professorship, University at Buffalo, SUNY 700 Ellicott Street, Buffalo, NY 14203-1102 hwi.buffalo.edu Phone: (716) 898 8631 Fax: (716) 898 8660 Skype:eddie.snell Email: esn...@hwi.buffalo.edu Webpage: https://hwi.buffalo.edu/scientist-directory/snell/ [cid:image001.png@01D5C619.F8914650] Heisenberg was probably here! From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Wednesday, January 8, 2020 11:29 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein concentration for the initial crystallisation trials I usually set a partial screen, maybe 24-48 conditions. If less than half the wells have precipitate, double protein concentration. If most have precipitate, maybe reduce protein or halve concentration of screen reagents. I usually start at 10 mg/mL or so. You can conveniently change protein conc. by manipulating protein/screen volume ratio. __ Roger Rowlett On Wed, Jan 8, 2020, 11:16 AM Armando Albert mailto:xalb...@iqfr.csic.es>> wrote: Dear all, I was wondering how to guess the optimal protein concentration for the initial crystallisation trials. Is there any trick or assay other than the classic PCT from Hampton? Armando To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
Re: [ccp4bb] Protein concentration for the initial crystallisation trials
Dear Armando, in case you have enough material to spare, I would use a concentrator to create a saturated solution, i.e. concentrate until it precipitates. Measure the concentration of the saturated solution, i.e., supernatant without disturbing the solution with the precipitate. You can start crystallisation trials at 70-90% of this concentration. Best, Tim On Wednesday, January 8, 2020 5:16:31 PM CET Armando Albert wrote: > Dear all, > I was wondering how to guess the optimal protein concentration for the > initial crystallisation trials. Is there any trick or assay other than the > classic PCT from Hampton? Armando > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 -- -- Tim Gruene Head of the Centre for X-ray Structure Analysis Faculty of Chemistry University of Vienna Phone: +43-1-4277-70202 GPG Key ID = A46BEE1A To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 signature.asc Description: This is a digitally signed message part.
Re: [ccp4bb] Protein concentration for the initial crystallisation trials
I usually set a partial screen, maybe 24-48 conditions. If less than half the wells have precipitate, double protein concentration. If most have precipitate, maybe reduce protein or halve concentration of screen reagents. I usually start at 10 mg/mL or so. You can conveniently change protein conc. by manipulating protein/screen volume ratio. __ Roger Rowlett On Wed, Jan 8, 2020, 11:16 AM Armando Albert wrote: > Dear all, > I was wondering how to guess the optimal protein concentration for the > initial crystallisation trials. Is there any trick or assay other than the > classic PCT from Hampton? > Armando > > > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 > To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Protein concentration for the initial crystallisation trials
Dear all, I was wondering how to guess the optimal protein concentration for the initial crystallisation trials. Is there any trick or assay other than the classic PCT from Hampton? Armando To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Protein concentration form chromatograms
Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
Re: [ccp4bb] Protein concentration form chromatograms
Step1: visit Protparam tool and CP your sequence, scroll down until you find the extinction coefficient part. If OD280 is not close to 1 then make sure to take that into account in your chromatogram say it is 0.7 step2: look at your mAU's If your peak is 1000 mAU then you have 0.7 mg/ml in that fraction Step3: you are pretty lazy google for it Jürgen On Jan 15, 2014, at 10:09 AM, Karel Chaz karel.c...@gmail.commailto:karel.c...@gmail.com wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
Re: [ccp4bb] Protein concentration form chromatograms
Hi Karel - To add to what Jurgen said, a few points on the measurement of the protein peak from the chromatogram. - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) - If your peak is a strong one, watch out for non-linearity in the absorbance measurement - my Akta UV monitor doesn't give a reliable reading once the A280 goes above 1.8. This will cause you to underestimate your total protein amount. - You also need to apply a correction factor since your UV cell path length isn't 1 cm. You could look up what the path length is in the manual, but the easiest way to do this is to compare UV readings for a set of fractions from your FPLC monitor and a standard spectrophotometer. Figure out the ratio of the two (it'll be a simple whole number, probably 2 or 5, or maybe 1 if your UV monitor does the correction automatically), then put it on a post-it next to the UV monitor so you won't have to do this again. Now multiply your chromatogram's integrated peak area by this factor to give you the standard (1 cm path length) A280 measurement. Hope that helps, Matt On 1/15/14 10:09 AM, Karel Chaz wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
Re: [ccp4bb] Protein concentration form chromatograms
On 1/15/14, 9:34 AM, Matthew Franklin wrote: - I usually approximate the peak as a triangle, so that the total peak area is 1/2 height (absorbance maximum) x base (the number of ml in your pool) GE's UNICORN for AKTAs (and probably the new Biorad FPLC software) integrates and tells you the area under whatever peak. Baselines and peak boundaries are user adjustable.
Re: [ccp4bb] Protein concentration form chromatograms
I am not sure, but probably path length not 1 cm in a detector cuvette . You must refer to your HPLS system manual for this value 15.01.2014 19:09, Karel Chaz пишет: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Protein concentration form chromatograms
Thanks for all the replies. And I would not call this laziness, rather crowdsourcing I should have added a few more details; it is easy to export from say Unicorn to excel, a list of pairs of values, Abs280 vs elution times, with which one can recreate the chromatogram. I wanted to use these for my calculations. K On Wed, Jan 15, 2014 at 10:49 AM, Evgeny Osipov e.m.osi...@gmail.comwrote: I am not sure, but probably path length not 1 cm in a detector cuvette . You must refer to your HPLS system manual for this value 15.01.2014 19:09, Karel Chaz пишет: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Protein concentration form chromatograms
I don't think it possible for protein purification purpose. There would be no linear correlation between A280 and the protein concentration. For pure protein analysis, as for minor amount protein used, it is possible. Acoot From: Karel Chaz karel.c...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, 15 January 2014 11:09 PM Subject: [ccp4bb] Protein concentration form chromatograms Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K
Re: [ccp4bb] Protein concentration form chromatograms
If using Unicorn, 1)Open your chromatogram in Evaluation window. 2)Go to Operations- Pool 3)Choose which baseline estimation suits you, define the pools (numbered rulers that appear under the chromatogram) 4)type in the path length (2 or 10 mm for UV-900 and 2 mm for UPC-900) 5)type in the mass extinction coefficient (Molar extinction coefficient/Mw in daltons) 6) get your answer from the table at the bottom of the screen The reading will make sense for pure protein. Also note that for the most accurate result you need to know the exact path length which varies a little bit from cell to cell (or so they say). Regards, Dmitry On 2014-01-15, at 10:09 AM, Karel Chaz wrote: Dear all, A question for the biochemistry-inclined folks in the bb; how do I calculate protein concentration of chromatography fractions, starting from Abs280 from the UV monitor? I know I could figure it out myself if I really tried, but why bother when I have access to so many brilliant minds Thanks to all, K smime.p7s Description: S/MIME cryptographic signature
Re: [ccp4bb] Protein concentration for crystallization
H..the real message from figure 3. The protein concentration. seems to be that 70% of proteins do NOT crystallize in the 10-12.5 mg bin, i.e. the 'right' concentration is an individual protein-in-that-buffer property - and all one can say is that the concentration needs to be high enough so that the crystallization conditions (either right away in case of batch, or vapor-diffusion assisted) can drive the system across the solubility limit into supersaturation. Most people actually working with their protein have already a reasonably developed idea what their protein can take in various buffer systems (valuable parameter!) . once they have scraped if off a centricon membrane, for example. The pre-screening originated from structural genomics when one had no idea what the 'customer' actually sent to the facility. If one knows the material it might be more efficient (i.e. more information from the same precious material) to set up a plate of 96 20+20 nl nanodrops than spending 2 ul on prescreening. Just an opinion sans statistics, BR From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debasish Chattopadhyay Sent: Tuesday, June 11, 2013 1:02 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein concentration for crystallization Perhaps my question was not expressed well. I wanted to know if proteins crystallize more frequently when the protein concentration is in the range 5-30mg/ml. The answer pointed out by my colleague Todd Green is on the page http://www.douglas.co.uk/PDB_data.htm Thanks for your inputs. Debasish From: Orru, Roberto [mailto:roberto.o...@emory.edu] Sent: Monday, June 10, 2013 5:04 PM To: Debasish Chattopadhyay Subject: RE: Protein concentration for crystallization Dear Debasish, On my memory there are 2 way (but I cannot say that are the only 2!) First: if you have the structure and you know the water content, you can guess the amount of protein crystallized in your drop by calculating the volume of the crystals. Second (if you can waste your drops): Fish all the crytsals in any drop for a given concentration, load a sds page w/ silver staining developing and compare it with a calibration curve done with your same protein in the same gel. Best R. _ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Debasish Chattopadhyay [debas...@uab.edu] Sent: Monday, June 10, 2013 10:49 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein concentration for crystallization What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg? Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480 _ This e-mail message (including any attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this message (including any attachments) is strictly prohibited. If you have received this message in error, please contact the sender by reply e-mail message and destroy all copies of the original message (including attachments).
Re: [ccp4bb] Protein concentration for crystallization
Bernhard, I'm often amazed at how forgiving protein crystallization is, or to put it another way, how efficient screens are at picking up crystallization hits even when you do everything wrong. However if you do go down to 20 + 20 nl drops you have to remember that probably 3/4 of your protein will be lost by sticking to the plastic of the plate or forming a skin at the air interface. You generally get roughly the same number of hits from 1 + 1 ul drops (where most of the protein is still in solution) as from 100 + 100 nl drops (roughly half the protein is lost), although the hits will be in different places. This implies that, within broad limits, things like protein concentration don't matter too much. My view of crystallization is that there's a group of proteins (lysozyme and many others) where all you have to do is gently push the protein out of solution, while taking care of nucleation (think microseeding). For these proteins it's a matter of *not *adding something that interferes with crystallization. Then there are others where you have to add something to the mix that stabilizes the crystal lattice. A smaller group requires 2 additives, a few may need 3 At the end there's a bunch that will never crystallize no matter what you do. It seems that protein concentration is of secondary importance in screening - although I accept that it may be crucial in optimization. Patrick On 11 June 2013 08:48, Bernhard Rupp hofkristall...@gmail.com wrote: H….the real message from figure 3. The protein concentration… seems to be that 70% of proteins do NOT crystallize in the 10-12.5 mg bin, i.e. the ‘right’ concentration is an individual protein-in-that-buffer property - and all one can say is that the concentration needs to be high enough so that the crystallization conditions (either right away in case of batch, or vapor-diffusion assisted) can drive the system across the solubility limit into supersaturation. Most people actually working with their protein have already a reasonably developed idea what their protein can take in various buffer systems (valuable parameter!) … once they have scraped if off a centricon membrane, for example… The pre-screening originated from structural genomics when one had no idea what the ‘customer’ actually sent to the facility. If one knows the material it might be more efficient (i.e. more information from the same precious material) to set up a plate of 96 20+20 nl nanodrops than spending 2 ul on prescreening. ** ** Just an opinion sans statistics, BR ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Debasish Chattopadhyay *Sent:* Tuesday, June 11, 2013 1:02 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] Protein concentration for crystallization ** ** Perhaps my question was not expressed well. I wanted to know if proteins crystallize more frequently when the protein concentration is in the range 5-30mg/ml. The answer pointed out by my colleague Todd Green is on the page http://www.douglas.co.uk/PDB_data.htm ** ** Thanks for your inputs. ** ** Debasish ** ** *From:* Orru, Roberto [mailto:roberto.o...@emory.eduroberto.o...@emory.edu] *Sent:* Monday, June 10, 2013 5:04 PM *To:* Debasish Chattopadhyay *Subject:* RE: Protein concentration for crystallization ** ** Dear Debasish, On my memory there are 2 way (but I cannot say that are the only 2!) First: if you have the structure and you know the water content, you can guess the amount of protein crystallized in your drop by calculating the volume of the crystals. Second (if you can waste your drops): Fish all the crytsals in any drop for a given concentration, load a sds page w/ silver staining developing and compare it with a calibration curve done with your same protein in the same gel. Best R. -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Debasish Chattopadhyay [debas...@uab.edu] *Sent:* Monday, June 10, 2013 10:49 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] Protein concentration for crystallization What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg? Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480 ** ** -- This e-mail message (including any attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this message (including any
[ccp4bb] Protein concentration for crystallization
What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg? Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480
Re: [ccp4bb] Protein concentration for crystallization
Dear Debasish, you can use REMARK 200 field in pdb file. Sadly, this field is not mandatory so not everyone provide protein concentration info. 10.06.2013 18:49, Debasish Chattopadhyay ?: What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg? Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480 -- Eugene Osipov Junior Research Scientist Laboratory of Enzyme Engineering A.N. Bach Institute of Biochemistry Russian Academy of Sciences Leninsky pr. 33, 119071 Moscow, Russia e-mail: e.m.osi...@gmail.com
Re: [ccp4bb] Protein concentration for crystallization
Dear Debasish What do you mean by percentage? do you mean consentration? so if you mean cons. I think you should test you protein using a TCP kit to observe at what cons. would your protein precipitate, this way you would verify the convinient cons. for your protein before crystallization Best Regards Rana From: Debasish Chattopadhyay debas...@uab.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, June 10, 2013 4:49 PM Subject: [ccp4bb] Protein concentration for crystallization What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg? Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480
Re: [ccp4bb] Protein concentration for crystallization
Perhaps my question was not expressed well. I wanted to know if proteins crystallize more frequently when the protein concentration is in the range 5-30mg/ml. The answer pointed out by my colleague Todd Green is on the page http://www.douglas.co.uk/PDB_data.htm Thanks for your inputs. Debasish From: Orru, Roberto [mailto:roberto.o...@emory.edu] Sent: Monday, June 10, 2013 5:04 PM To: Debasish Chattopadhyay Subject: RE: Protein concentration for crystallization Dear Debasish, On my memory there are 2 way (but I cannot say that are the only 2!) First: if you have the structure and you know the water content, you can guess the amount of protein crystallized in your drop by calculating the volume of the crystals. Second (if you can waste your drops): Fish all the crytsals in any drop for a given concentration, load a sds page w/ silver staining developing and compare it with a calibration curve done with your same protein in the same gel. Best R. From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Debasish Chattopadhyay [debas...@uab.edu] Sent: Monday, June 10, 2013 10:49 To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein concentration for crystallization What would be a convenient way to estimate what percentages of proteins have been crystallized in a concentration range, for example 5-30 mg? Debasish Chattopadhyay University of Alabama at Birmingham CBSE-250 1025 18th Street South, Birmingham, Al-35294 USA Ph: (205)934-0124; Fax: (205)934-0480 This e-mail message (including any attachments) is for the sole use of the intended recipient(s) and may contain confidential and privileged information. If the reader of this message is not the intended recipient, you are hereby notified that any dissemination, distribution or copying of this message (including any attachments) is strictly prohibited. If you have received this message in error, please contact the sender by reply e-mail message and destroy all copies of the original message (including attachments).
Re: [ccp4bb] Protein concentration vs Molecular wt...
Number of years ago Jaru Jancarik (the author of Screen I II sold by HR) while in Berkeley Structural Genomics Center (or may be even earlier) made an observation regarding protein precipitation in condition A6 in that very screen. Based on this observation HR sells now PCT (protein concentration test or something similar). In brief, if your protein is concentrated enough and is a regular protein, not a stellar, you will see medium to heavy precipitation. In the case of proteins that are really of great quality you'll end up having crystals in A6. After many years of experience I realized that A9 and A10 from the same screen could be added to the list of conditions that are somewhat indicative for choosing right concentration same way like Jaru did for A6. My two Armenian drams worth, Vaheh From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Thursday, July 19, 2012 4:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Protein concentration vs Molecular wt... This is almost exactly our basic approach, too. Before we got a dropsetter, we did 24 wells (1/2 screen) to get a feel for the correct protein concentration. Some additional rules of thumb we use: 1. We usually start at 10 mg/mL protein and go up or down from there depending on the results of the initial screen 2. If we observe a high frequency of precipitation in a 10 mg/mL protein screen, we will usually set a 1/2 concentration screen by diluting the screen solutions 1:1 with water. This frequently uncovers additional hits in wells that were heavily precipitated in the original screen. Empirically, proteins we study seem to crystallize better in the higher protein/lower precipitant zone of the phase diagram than the lower protein/higher precipitant zone. YMMV. 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.edumailto:rrowl...@colgate.edu On 7/19/2012 4:31 PM, mjvdwo...@netscape.netmailto:mjvdwo...@netscape.net wrote: I don't think there is such a rule, but in the old days, when we only had Hampton Screen I and II, the rule was: - Set up screen 1, look at the drops and you should expect some kind of precipitation in 50% of the drops. If much less than that, increase your protein concentration. If much more than that, decrease protein concentration. - Set up screen 2, look and expect 30% precipitation. I used to cut corners and do the statistics at 1/2 of a screen (one 24-well plate). You can probably use this method to get within a factor of 2 of the optimal concentration. There are probably good statistics in the papers for the screens that you may use. One of the advantages of structural genomics efforts is that these things are known (and hopefully published). Even older trick is to take a drop of protein and look under a microscope, record how much AmSO4 it takes to cause precipitation. Do the same with PEG. Keep adding a little at a time and look immediately. This will give you an idea if you are near a reasonable concentration. I think that this latter method does not tell you much more than physics-information - which is how many zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable. Mark -Original Message- From: james09 pruza james09x...@gmail.commailto:james09x...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Sent: Thu, Jul 19, 2012 1:59 pm Subject: [ccp4bb] Protein concentration vs Molecular wt... Dear Crystallographers, Is there any rule of thumb for Protein concentration and molecular weight for crystallization trials of a soluble protein? Looking for high molecular wt. protein ~50kDa. James. To the extent this electronic communication or any of its attachments contain information that is not in the public domain, such information is considered by MedImmune to be confidential and proprietary. This communication is expected to be read and/or used only by the individual(s) for whom it is intended. If you have received this electronic communication in error, please reply to the sender advising of the error in transmission and delete the original message and any accompanying documents from your system immediately, without copying, reviewing or otherwise using them for any purpose. Thank you for your cooperation.
[ccp4bb] Protein concentration vs Molecular wt...
Dear Crystallographers, Is there any rule of thumb for Protein concentration and molecular weight for crystallization trials of a soluble protein? Looking for high molecular wt. protein ~50kDa. James.
Re: [ccp4bb] Protein concentration vs Molecular wt...
I don't think there is such a rule, but in the old days, when we only had Hampton Screen I and II, the rule was: - Set up screen 1, look at the drops and you should expect some kind of precipitation in 50% of the drops. If much less than that, increase your protein concentration. If much more than that, decrease protein concentration. - Set up screen 2, look and expect 30% precipitation. I used to cut corners and do the statistics at 1/2 of a screen (one 24-well plate). You can probably use this method to get within a factor of 2 of the optimal concentration. There are probably good statistics in the papers for the screens that you may use. One of the advantages of structural genomics efforts is that these things are known (and hopefully published). Even older trick is to take a drop of protein and look under a microscope, record how much AmSO4 it takes to cause precipitation. Do the same with PEG. Keep adding a little at a time and look immediately. This will give you an idea if you are near a reasonable concentration. I think that this latter method does not tell you much more than physics-information - which is how many zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable. Mark -Original Message- From: james09 pruza james09x...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Jul 19, 2012 1:59 pm Subject: [ccp4bb] Protein concentration vs Molecular wt... Dear Crystallographers, Is there any rule of thumb for Protein concentration and molecular weight for crystallization trials of a soluble protein? Looking for high molecular wt. protein ~50kDa. James.
Re: [ccp4bb] Protein concentration vs Molecular wt...
This is almost exactly our basic approach, too. Before we got a dropsetter, we did 24 wells (1/2 screen) to get a feel for the correct protein concentration. Some additional rules of thumb we use: 1. We usually start at 10 mg/mL protein and go up or down from there depending on the results of the initial screen 2. If we observe a high frequency of precipitation in a 10 mg/mL protein screen, we will usually set a 1/2 concentration screen by diluting the screen solutions 1:1 with water. This frequently uncovers additional hits in wells that were heavily precipitated in the original screen. Empirically, proteins we study seem to crystallize better in the higher protein/lower precipitant zone of the phase diagram than the lower protein/higher precipitant zone. YMMV. 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 7/19/2012 4:31 PM, mjvdwo...@netscape.net wrote: I don't think there is such a rule, but in the old days, when we only had Hampton Screen I and II, the rule was: - Set up screen 1, look at the drops and you should expect some kind of precipitation in 50% of the drops. If much less than that, increase your protein concentration. If much more than that, decrease protein concentration. - Set up screen 2, look and expect 30% precipitation. I used to cut corners and do the statistics at 1/2 of a screen (one 24-well plate). You can probably use this method to get within a factor of 2 of the optimal concentration. There are probably good statistics in the papers for the screens that you may use. One of the advantages of structural genomics efforts is that these things are known (and hopefully published). Even older trick is to take a drop of protein and look under a microscope, record how much AmSO4 it takes to cause precipitation. Do the same with PEG. Keep adding a little at a time and look immediately. This will give you an idea if you are near a reasonable concentration. I think that this latter method does not tell you much more than physics-information - which is how many zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable. Mark -Original Message- From: james09 pruza james09x...@gmail.com To: CCP4BB CCP4BB@JISCMAIL.AC.UK Sent: Thu, Jul 19, 2012 1:59 pm Subject: [ccp4bb] Protein concentration vs Molecular wt... Dear Crystallographers, Is there any rule of thumb for Protein concentration and molecular weight for crystallization trials of a soluble protein? Looking for high molecular wt. protein ~50kDa. James.
Re: [ccp4bb] Protein concentration vs Molecular wt...
Hi James, You can get the PCT (Pre-Crystallisation Test) from one of the more famous manufacturers of crystallography products - essentially you can quickly screen your protein to get an idea if it is too dilute, too concentrated, or somewhere in the middle. Of course, the results are representative only (I've had samples which looked great on the PCT but then gave me ~90% clear drops even at 40 mg/ml, and an 80 kDa protein which crystallised at 2 mg/ml) so as with anything they should be taken with the appropriate vessel full of sodium chloride... If you have enough protein, then I agree with the previous comments that it is more useful to set up some trays and see what % of conditions give hits, and work from there. Cheers, Tom On 19 July 2012 20:58, james09 pruza james09x...@gmail.com wrote: Dear Crystallographers, Is there any rule of thumb for Protein concentration and molecular weight for crystallization trials of a soluble protein? Looking for high molecular wt. protein ~50kDa. James. -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
[ccp4bb] protein concentration in crystal
Hi all I'm looking for a way to calculate the protein concentration in a single crystal. So what I'm thinking is to use the Matthew's number to calculate how many molecules inside the crystal, then multiply that number by Avogadro's number to get moles then divide by volume of crystal. Is this approach correct? Thank you for your advice/suggestion Happy holidays! :) Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 If you never did you should. These things are fun and fun is good Dr. Seuss
Re: [ccp4bb] protein concentration in crystal
Sounds like a good units conversion problem for Introductory Chemistry. Basically, you can get molecules per cubic angstrom from your unit cell analysis (molecules per unit cell and cubic angstroms per unit cell) and convert to the units of your choice, mg/cm^3, mol/L (actually mol/dm^3 in this case), or whatever. Or you can use your Matthews number and start from A^3/Dalton. Cheers. On 12/13/2010 3:07 PM, Teresa De la Mora wrote: Hi all I'm looking for a way to calculate the protein concentration in a single crystal. So what I'm thinking is to use theMatthew's number to calculate how many molecules inside the crystal, then multiply that number by Avogadro's number to get moles then divide by volume of crystal. Is this approach correct? Thank you for your advice/suggestion Happy holidays! :) Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 "If you never did you should. These things are fun and fun is good" Dr. Seuss -- Roger S. Rowlett 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
Re: [ccp4bb] protein concentration in crystal
Specific density of protein is about 1.34 g/cm3. Correct by solvent fraction. About 600-700mg/ml for 50% for example. Compute molarity from there. BR From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Teresa De la Mora Sent: Monday, December 13, 2010 12:08 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein concentration in crystal Hi all I'm looking for a way to calculate the protein concentration in a single crystal. So what I'm thinking is to use the Matthew's number to calculate how many molecules inside the crystal, then multiply that number by Avogadro's number to get moles then divide by volume of crystal. Is this approach correct? Thank you for your advice/suggestion Happy holidays! :) Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 If you never did you should. These things are fun and fun is good Dr. Seuss
Re: [ccp4bb] protein concentration in crystal
If you know the Matthews volume, then you can convert to protein concentration with: [protein] (mg/mL) = 1/( V_M (A^3/Da) ) * 1.66e-21 (mg/Da) * 1e24 (A^3/mL) For example, lysozyme crystals (V_M = 2.0 A^3/Da) contain about 830 mg/ml of protein. Most protein crystals are in this ballpark (because V_M doesn't have much of a range). If you want molarity, then you also need to know the molecular weight (MW): [protein] (mol/L) = [protein] (mg/mL) / MW (g/mol) Or about 60 mM lysozyme monomers in a crystal. -James Holton MAD Scientist On 12/13/2010 12:07 PM, Teresa De la Mora wrote: Hi all I'm looking for a way to calculate the protein concentration in a single crystal. So what I'm thinking is to use the Matthew's number to calculate how many molecules inside the crystal, then multiply that number by Avogadro's number to get moles then divide by volume of crystal. Is this approach correct? Thank you for your advice/suggestion Happy holidays! :) Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 If you never did you should. These things are fun and fun is good Dr. Seuss
Re: [ccp4bb] protein concentration in crystal
Hello Teresa, c = x/(N.V) where x=number of molecules per unit cell, N=Avogadro's number, V=volume of the unit cell in liter, c=concentration in molar Cheers Filip On Mon, Dec 13, 2010 at 12:07 PM, Teresa De la Mora dela0...@umn.eduwrote: Hi all I'm looking for a way to calculate the protein concentration in a single crystal. So what I'm thinking is to use the Matthew's number to calculate how many molecules inside the crystal, then multiply that number by Avogadro's number to get moles then divide by volume of crystal. Is this approach correct? Thank you for your advice/suggestion Happy holidays! :) Teresa Teresa De la Mora-Rey Ph.D. Dept. Medicinal Chemistry University of Minnesota 8-101 Weaver-Densford Hall 308 Harvard St. SE, Minneapolis, MN 55455 Lab phone (612) 626-5226 If you never did you should. These things are fun and fun is good Dr. Seuss -- Filip Van Petegem, PhD Assistant Professor The University of British Columbia Dept. of Biochemistry and Molecular Biology 2350 Health Sciences Mall - Rm 2.356 Vancouver, V6T 1Z3 phone: +1 604 827 4267 email: filip.vanpete...@gmail.com http://crg.ubc.ca/VanPetegem/
[ccp4bb] Protein concentration
Dear all, I would be glad to hear what (simple) method I should use to determine protein concentrations as accurately as possible. Presently, I'm measuring absorption at 280nm with a nanodrop device. I either have 0 or 1 tryptophan and no activity test. Many thanks in advance! Best regards, Mark Mark Hilge Protein Biophysics NCMLS 274 3rd floor M850.03.035 Geert Grooteplein 28 6525 GA Nijmegen The Netherlands http://www.mark-hilge.com Phone: 0031 24 36 10 525 Het UMC St Radboud staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud University Nijmegen Medical Centre is listed in the Commercial Register of the Chamber of Commerce under file number 41055629.
Re: [ccp4bb] Protein concentration
Dear Mark Most easy way to have relative estimation is to run PAGE of your protein with known proteins of known different concentration so u can have a good relative estimation. but for more accurate estimation u can go for Biochemical methods such as Lowry method or BCA method which are more senstive then UV spectro method although they depend upon oxidation of total aromatic amino acid in protein u can try or wait for other suggestions.# . Puneet Juneja On Thu, Aug 21, 2008 at 12:54 PM, Mark Hilge [EMAIL PROTECTED] wrote: Dear all, I would be glad to hear what (simple) method I should use to determine protein concentrations as accurately as possible. Presently, I'm measuring absorption at 280nm with a nanodrop device. I either have 0 or 1 tryptophan and no activity test. Many thanks in advance! Best regards, Mark Mark Hilge Protein Biophysics NCMLS 274 3rd floor M850.03.035 Geert Grooteplein 28 6525 GA Nijmegen The Netherlands http://www.mark-hilge.com Phone: 0031 24 36 10 525 Het UMC St Radboud staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud University Nijmegen Medical Centre is listed in the Commercial Register of the Chamber of Commerce under file number 41055629.
Re: [ccp4bb] Protein concentration
Often the exact concentration is not so important compared to the ability to establish proportional read-out, ease and reproducibility so that systematic variations and comparisons can be made. For considerations of molar ratios of for example protein:ligand complexes one would often screen a range or add in excess in any case, so probably again you are fine. For an accurate calibration of your method of choice you can opt for a total aminoacid analysis. Poul On 21/08/2008, at 15.07, Puneet juneja wrote: Dear Mark Most easy way to have relative estimation is to run PAGE of your protein with known proteins of known different concentration so u can have a good relative estimation. but for more accurate estimation u can go for Biochemical methods such as Lowry method or BCA method which are more senstive then UV spectro method although they depend upon oxidation of total aromatic amino acid in protein u can try or wait for other suggestions.# . Puneet Juneja On Thu, Aug 21, 2008 at 12:54 PM, Mark Hilge [EMAIL PROTECTED] wrote: Dear all, I would be glad to hear what (simple) method I should use to determine protein concentrations as accurately as possible. Presently, I'm measuring absorption at 280nm with a nanodrop device. I either have 0 or 1 tryptophan and no activity test. Many thanks in advance! Best regards, Mark Mark Hilge Protein Biophysics NCMLS 274 3rd floor M850.03.035 Geert Grooteplein 28 6525 GA Nijmegen The Netherlands http://www.mark-hilge.com Phone: 0031 24 36 10 525 Het UMC St Radboud staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud University Nijmegen Medical Centre is listed in the Commercial Register of the Chamber of Commerce under file number 41055629.
Re: [ccp4bb] Protein concentration
Mark, A little more information on the protein and need would be nice. Is it a large peptide, a small protein, or a recombinant protein? Do you want real quantitative results or semi quantitative (like BCA, Bradford, or Lowry which can be off by 20% or more relative to the [BSA])? Do you want it to be rapid assay? Is it to measure the protein concentration of a pure sample or to follow a purification? Do you want it to be non-invasive (like the A280 measurement) or how much protein are you willing to waste? Some thoughts for a pure sample: 1) If you denature a sample, and label the lysines and N-terminus with a fluorophore (e.g., fluoroscein), you can unambiguously measure the absorption or fluouresence emission. But that a longish procedure and you lose your protein. If you have Cys residues, labeling those with DTNB or mercurichrome can allow you to do the same thing. You just need to ensure all labeling sites are accessible. 2) Many people who recombinantly express a protein without Trp residues have mutated a Phe or Tyr residue to Trp. Thus, the protein has a built-in means to measure protein concentration by Trp absorption or fluorescence. 3) If you have protein to waste or it survives being lyophilized, dialyze it into ammonium acetate, lyophilize it, then weigh it on a microbalance. Measuring protein concentration is not an exact science if you want it to be rapid and not waste lots of your precious protein sample. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Thu, Aug 21, 2008 at 12:54 PM, Mark Hilge [EMAIL PROTECTED] wrote: Dear all, I would be glad to hear what (simple) method I should use to determine protein concentrations as accurately as possible. Presently, I'm measuring absorption at 280nm with a nanodrop device. I either have 0 or 1 tryptophan and no activity test. Many thanks in advance! Best regards, Mark Mark Hilge Protein Biophysics NCMLS 274 3rd floor M850.03.035 Geert Grooteplein 28 6525 GA Nijmegen The Netherlands http://www.mark-hilge.com Phone: 0031 24 36 10 525 Het UMC St Radboud staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud University Nijmegen Medical Centre is listed in the Commercial Register of the Chamber of Commerce under file number 41055629.
Re: [ccp4bb] Protein concentration
Mark, There are two very easy alternatives. First is the Bradford Coomassie Dye binding assay. You can buy a kit from Biorad at the following link: http://www.bio-rad.com/B2B/BioRad/product/br_category.jsp?BV_SessionID=0349900713.1219329576BV_EngineID=ccchadeemgdfflfcfngcfkmdhkkdfll.0categoryPath=%2fCatalogs%2fLife+Science+Research%2fSample+Quantitation%2fProtein+Assay+Kits+and+Cuvettes%2fBio-Rad+Protein+AssaydivName=CorporateloggedIn=falselang=Englishcountry=HQcatLevel=5catOID=-12777isPA=falseserviceLevel=Lit+RequestsearchStr=coomassiecateName=Ordering+Information Second is the BCA assay which can be purchased from Pierce (now bought out by Thermo/Fisher): http://www.piercenet.com/Products/Browse.cfm?fldID=02020101 Cheers, Jim On Thu, Aug 21, 2008 at 6:54 AM, Mark Hilge [EMAIL PROTECTED] wrote: Dear all, I would be glad to hear what (simple) method I should use to determine protein concentrations as accurately as possible. Presently, I'm measuring absorption at 280nm with a nanodrop device. I either have 0 or 1 tryptophan and no activity test. Many thanks in advance! Best regards, Mark Mark Hilge Protein Biophysics NCMLS 274 3rd floor M850.03.035 Geert Grooteplein 28 6525 GA Nijmegen The Netherlands http://www.mark-hilge.com Phone: 0031 24 36 10 525 Het UMC St Radboud staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud University Nijmegen Medical Centre is listed in the Commercial Register of the Chamber of Commerce under file number 41055629. -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 [EMAIL PROTECTED] [EMAIL PROTECTED]
