Re: [ccp4bb] methods to capture proteins from cell culture medium
Bei, How do you concentrate your media? We use tangential flow filtration. If you get a good filter (Millipore Spiral wound TFF is one example) it goes pretty quick, in ~2 hours you should be able to process 4L(concentrate+ dilute several time in buffer). We secrete proteins from insect cells, but something in the media will strip the Ni2+ from our IMAC column if we apply the sup directly. I have tried AS precipitation and peg precipitation from culture media. Both worked fine at the small scale, but when scaling up I ran into 2 issues: 1. You have to add huge amounts of AS (like kilograms), which increases your volume a good amount, 2. my 'pellets' would actually float on top of the media after spinning, which was tough to deal with. I have also tried loading the media onto a large Q column, but that didn't work well for me-fractions were too messy. I think you best option is to get a good TFF setup, do your concentration/buffer exchange, and go right to your IMAC column Nat Nat Clark Graduate Student Garman Lab Biochemistry and Molecular Biology Dept. UMass Amherst 2011/4/13 joybeiyang joybeiy...@gmail.com: Dear all, Thanks a lot for sharing, seems that either a HIC column or AS would work, and that's great, I should give both of them a try. I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Thank you very much again! Bei 2011-04-12 joybeiyang 发件人： mi...@chem.ucla.edu 发送时间： 2011-04-12 18:34:27 收件人： joybeiyang 抄送： CCP4BB@JISCMAIL.AC.UK 主题： Re: [ccp4bb] methods to capture proteins from cell culture medium Bei, I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column. Good luck, Mike - Original Message - From: joybeiyang joybeiy...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] methods to capture proteins from cell culture medium
Hi Bei, For the extracellular protein I worked on in graduate school, I typically purified it from 4 L preps in LB media. The standard protocol was to do a crude low cut with ammonium sulfate cut followed by precipitation of the protein with a high cut. The pellet was then resuspended, dialyzed and loaded on an S-sepharose column. I experimented with taking the media (after spinning out the cells) and diluting 1:1 with a low ionic strength buffer and loading directly onto the S-sepharose column. This worked, but the loading time for 8 L was so long it was not worth it. Another option might have been to use bulk media to bind the protein in a batch step. I never tested this. A final option that we explored was Expanded-Bed Adsorption Chromatography. This would allow us to get rid of the initial centrifugation step to remove the cells from the media and would allow fast loading with a high speed pump. We priced out the media, column and pump from Pharmacia at the time, but never ended up purchasing the system. The technology looked promising and should have worked well for our system, but we decided that we did not need to do too many more preps for the project and just used the standard protocol. Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of joybeiyang Sent: Tuesday, April 12, 2011 2:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang
[ccp4bb] methods to capture proteins from cell culture medium
Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang
Re: [ccp4bb] methods to capture proteins from cell culture medium
Bei, I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column. Good luck, Mike - Original Message - From: joybeiyang joybeiy...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] methods to capture proteins from cell culture medium
Dear all, Thanks a lot for sharing, seems that either a HIC column or AS would work, and that's great, I should give both of them a try. I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Thank you very much again! Bei 2011-04-12 joybeiyang 发件人： mi...@chem.ucla.edu 发送时间： 2011-04-12 18:34:27 收件人： joybeiyang 抄送： CCP4BB@JISCMAIL.AC.UK 主题： Re: [ccp4bb] methods to capture proteins from cell culture medium Bei, I had a former labmate who had the same situation and would load somewhere between 6-8L of media directly onto a column. I don't remember what type of column it was, ion exchange may not be ideal if the ionic strength of your medium is high. I think it may have been a phenyl sepharose column. Good luck, Mike - Original Message - From: joybeiyang joybeiy...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, April 12, 2011 2:13:49 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] methods to capture proteins from cell culture medium Dear all, My protein of interest was expressed as secreted protein, so I have to collect the medium and change the buffer with sortorius Jet before I load the sample onto a IMAC, the buffer change step in my current protocol can last for 12hrs (I have to concentrate 4L to 200ml, then dilute it with lysis buffer and concentrate it again, then dilute and concentrate repeatedly) and is really boring and troublesome, besides I always observe protein loss during this step and the detergent in the medium usually concentrate as well in this step which would interfere with subsequent purification process. I am wondering if there are more convenient ways to capture the target protein from medium? How about the following: 1. directly load the medium onto a ion exchange column? 2. Amonium sulfate precipitation? 3. anyother thoughts? Thank you very much in advance! Best, Bei 2011-04-12 joybeiyang -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] methods to capture proteins from cell culture medium
I thought about HIC too, but do not know if it would work since the binding of protein to HIC need high salt conc. and I am not sure if the salt conc. in the sf900 or Hi5 medium is high enough (the formulation is secret, LOL), thus it is good to know that someone has succesful experience with HIC. Few things: 1. With regard to the salt/ionic strength, the formulation of serum-free media cannot be far off from the traditional media. Very crudely speaking, figure an equivalent of ~200 mM NaCl. So most protein won't bind to, say, phenyl sepharose under these conditions. But your protein might be in the minority - who knows? 2. What prevents you from adding extra salt to the collected medium? Say, ~1 M ammonium sulphate final? There probably will be some precipitate forming which can be filtered away before loading onto a HIC column. 3. Hydroxylapatite. Ceramic Type I version from Bio-Rad in particular. Large size beads packed into a wide column. A great way to concentrate total protein at high flow rates. Phosphate concentration in the medium is low enough that majority of proteins will sill bind. - Dima
