Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
If you want to just reduce the volume/concentrate proteins, you can use Millipore's Stirred cell (high-output stirred cell, 2,000 ml capacity). Zhongren Wu From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Xuan Yang Sent: Thursday, September 03, 2009 5:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
may be diafiltration devices might work for you. the membranes are kind of expensive but we had used this effectively to reduce the volume of cell culture media where we had secreted proteins in large volumes of culture media. here is a link http://www.spectrapor.com/lit/hfdial.pdf padayatti On Sun, Sep 6, 2009 at 10:16 PM, Wu, Zhongrenz...@vitaerx.com wrote: If you want to just reduce the volume/concentrate proteins, you can use Millipore’s Stirred cell (high-output stirred cell, 2,000 ml capacity). Zhongren Wu From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Xuan Yang Sent: Thursday, September 03, 2009 5:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang -- Pius S Padayatti Scientist, Polgenix, Inc. 11000 Cedar Ave, Suite 260 Cleveland, OH 44106 Phone: 216-658-4528 Fax: 216-658-4529
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
may be diafiltration devices might work for you. the membranes are kind of expensive but we had used this effectively to reduce the volume of cell culture media where we had secreted proteins in large volumes of culture media. here is a link http://www.spectrapor.com/lit/hfdial.pdf padayatti On Thu, Sep 3, 2009 at 3:52 AM, Xuan Yangpattisy...@gmail.com wrote: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang -- Pius S Padayatti Scientist, Polgenix, Inc. 11000 Cedar Ave, Suite 260 Cleveland, OH 44106 Phone: 216-658-4528 Fax: 216-658-4529
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Hi, Could you not carry out Ammonium Sulfate precipitation to concentrate the protein? I never did it myself so I have no idea if it's practical in this case. Maybe it's still worth mentioning, though. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Fri, 4 Sep 2009, Radovan Spurny wrote: Stirred cell from Milipore is very useful for concentrating big volumes of protein solution. There are cells from 3 to 400 mL. Ultrafiltration is performed through the membrane with high pressure by connecting to inert gas (nitrogen). It is very fast and comfortable technique. Here are more information: http://www.millipore.com/catalogue/module/C3259 Sincerely, Radovan Spurny, PhD Laboratory of Structural Neurobiology Division of Pharmacology Campus Gasthuisberg, ON1 Herestraat 49, PB 601 B-3000 Leuven Belgium e-mail: radovan.spu...@med.kuleuven.be From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Yingyun Liu [yingyun...@jhu.edu] Sent: Thursday, September 03, 2009 16:48 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale An anion exchange column with a large enough bed volume might be used to concentrate the protein. You probably need to change the pH of the solution so that the protein sticks to the column. On Thu, 2009-09-03 at 17:05 +0800, Xuan Yang wrote: Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Stirred cell from Milipore is very useful for concentrating big volumes of protein solution. There are cells from 3 to 400 mL. Ultrafiltration is performed through the membrane with high pressure by connecting to inert gas (nitrogen). It is very fast and comfortable technique. Here are more information: http://www.millipore.com/catalogue/module/C3259 Sincerely, Radovan Spurny, PhD Laboratory of Structural Neurobiology Division of Pharmacology Campus Gasthuisberg, ON1 Herestraat 49, PB 601 B-3000 Leuven Belgium e-mail: radovan.spu...@med.kuleuven.be From: CCP4 bulletin board [ccp...@jiscmail.ac.uk] On Behalf Of Yingyun Liu [yingyun...@jhu.edu] Sent: Thursday, September 03, 2009 16:48 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale An anion exchange column with a large enough bed volume might be used to concentrate the protein. You probably need to change the pH of the solution so that the protein sticks to the column. On Thu, 2009-09-03 at 17:05 +0800, Xuan Yang wrote: Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
Dear Xuan Yang, for such large volumes we use a Pellicon type system. The one we have in the lab is a polyethersulfone PTJK prep/Scale TFF 2.5 FT2 High flux 10 k from Millipore. good luck Demetres Xuan Yang wrote: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang -- Demetres D. Leonidas, Ph.D. Senior Researcher Structural Biology Chemistry Group Institute of Organic and Pharmaceutical Chemistry The National Hellenic Research Foundation 48, Vassileos Constantinou Avenue Athens 116 35, Greece == Tel. +30 210 7273841 (office) +30 210 7273895 (lab) Fax. +30 210 7273831 E-mail: d...@eie.gr URL: http://athena.eie.gr ==
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
An anion exchange column with a large enough bed volume might be used to concentrate the protein. You probably need to change the pH of the solution so that the protein sticks to the column. On Thu, 2009-09-03 at 17:05 +0800, Xuan Yang wrote: Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
Re: [ccp4bb] Devices Suitable to Concentrate Protein Solutions in Liter Scale
1. Yes, Ion Exchange (not just Anion as we don't know anything about their protein (unless I missed an email)) is a great capture step if you have the time to mess about. 2. When I have been refolding recently, after the refolding step we have found that a good, old-fashioned Ammonium sulphate cut step (well, two of them) has worked really, really well. It takes a bit of tinkering, but it really does work a treat. Email me if you want to know about inner design of such an experiment. We are working with an untagged version of the protein as we use yet another affinity step that doesn't require it... see below. 3. If you know something about your protein, you might be able to use a different affinity step to cature it also. Heparin - Your protein may interact with a heparin column, even if you have no reason to think it will. Blue HP - Actually, the only reason I mention this one is that it has worked so well for one of the proteins I am working on at the mo. GE reckon if your protein has an affinity for nucleotides, nicotinamides blah blah blah it might interact with your protein. This can be a really powerful capture column if it does. 4. A great tag to capture it would be a Z-tag. cheers charlie An anion exchange column with a large enough bed volume might be used to concentrate the protein. You probably need to change the pH of the solution so that the protein sticks to the column. On Thu, 2009-09-03 at 17:05 +0800, Xuan Yang wrote: Dear Dr Ku, It was a wonderful idea! However, the refolding buffer contained 500mM L-Arg and 1mM EDTA. And I want to try other affinity columns, just don't know what resin would be appropriate. Sincerely, Xuan Yang 2009/9/3 Shao-Yang Ku s...@embl-hamburg.de Can you put a 6His-tag on your protein and add some Ni-NTA beads to your solution to capture (and concentrate) the properly folded molecules? Quoting Xuan Yang pattisy...@gmail.com: Dear All, I am working on protein refolding via dialysis in large volumn (typically 2~4 litters). It was problematic when I wanted to concentrate the solution to at least less than 500ml. If you know any device appropriate for such task, please help me out:) Thanks in advance! Sincerely, Xuan Yang
