Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Dear Zhen, I should also point out that the statement Matt made (Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins.) is not completely correct. Superdex and all chromatographic media made from carbohydrates (dextran, agarose, etc.) are also quite hydrophobic (which is surprising to some). The observation Zhen made is the classic behavior of hydrophobic interaction with the gel filtration media, which led to the development of hydrophobic interaction chromatographic (HIC). For HIC, you load the protein in high salt, and elute with low salt or a chaotrope (LiCl). So when you redo you experiment, Zhen, try with AND without salt. As Matt said, if it is an ion-exchange interaction, extra salt will make it elute more normally. But if it gets worse, then the extra salt is increasing the hydrophobic interactions, and you should run the column in lower salt (~50 mM NaCl) or with a bit of detergent. Given that you are refolding your protein, a partially folded protein may have have more hydrophobic patches. As my lab is routinely refolding our target proteins, we are always watching for this behavior, including on our analytical Superdex 200 10/300 columns, which is one of our favorites. Excessive hydrophobic interactions can also lead to clogged columns, which is not what you want for this expensive column. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On Jun 20, 2013, at 5:38 PM, Zhang, Zhen wrote: Hi Matthew, Thanks a lot. That is a great idea. I will try the high salt and worry about the crystallization later. Zhen -Original Message- From: Matthew Franklin [mailto:mfrank...@nysbc.org] Sent: Thursday, June 20, 2013 4:34 PM To: Zhang, Zhen Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Hi Zhen - Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins. This is why the manufacturer recommends including a certain amount of salt in the running buffer. I have had the same experience with a few proteins, including one that came off the column well after the salt peak! (The protein was very clean after this step; all other proteins had eluted earlier.) As others have said, you can't rely on molecular weight calibrations in this case, but this behavior alone is no reason to think that the protein is misfolded or otherwise badly behaved. If you don't like the late elution, try increasing the salt concentration of your running buffer to 250 or even 500 mM. You'll probably need to exchange the eluted protein back into a low-salt buffer for your next steps (e.g. crystallization) if you do this. - Matt On 6/20/13 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. -- 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] Puzzling observation about size exclusion chromatography
Hi Michael, Thank you very much for your suggestions. I will rerun with both buffer conditions and report back later. Thanks to everyone for your reply. Zhen From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R. M. Garavito Sent: Friday, June 21, 2013 9:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Dear Zhen, I should also point out that the statement Matt made (Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins.) is not completely correct. Superdex and all chromatographic media made from carbohydrates (dextran, agarose, etc.) are also quite hydrophobic (which is surprising to some). The observation Zhen made is the classic behavior of hydrophobic interaction with the gel filtration media, which led to the development of hydrophobic interaction chromatographic (HIC). For HIC, you load the protein in high salt, and elute with low salt or a chaotrope (LiCl). So when you redo you experiment, Zhen, try with AND without salt. As Matt said, if it is an ion-exchange interaction, extra salt will make it elute more normally. But if it gets worse, then the extra salt is increasing the hydrophobic interactions, and you should run the column in lower salt (~50 mM NaCl) or with a bit of detergent. Given that you are refolding your protein, a partially folded protein may have have more hydrophobic patches. As my lab is routinely refolding our target proteins, we are always watching for this behavior, including on our analytical Superdex 200 10/300 columns, which is one of our favorites. Excessive hydrophobic interactions can also lead to clogged columns, which is not what you want for this expensive column. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 603 Wilson Rd., Rm. 513 Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.commailto:garav...@gmail.com On Jun 20, 2013, at 5:38 PM, Zhang, Zhen wrote: Hi Matthew, Thanks a lot. That is a great idea. I will try the high salt and worry about the crystallization later. Zhen -Original Message- From: Matthew Franklin [mailto:mfrank...@nysbc.org] Sent: Thursday, June 20, 2013 4:34 PM To: Zhang, Zhen Cc: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Hi Zhen - Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins. This is why the manufacturer recommends including a certain amount of salt in the running buffer. I have had the same experience with a few proteins, including one that came off the column well after the salt peak! (The protein was very clean after this step; all other proteins had eluted earlier.) As others have said, you can't rely on molecular weight calibrations in this case, but this behavior alone is no reason to think that the protein is misfolded or otherwise badly behaved. If you don't like the late elution, try increasing the salt concentration of your running buffer to 250 or even 500 mM. You'll probably need to exchange the eluted protein back into a low-salt buffer for your next steps (e.g. crystallization) if you do this. - Matt On 6/20/13 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
[ccp4bb] Puzzling observation about size exclusion chromatography
Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type. Pat On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Zhen, I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other techniques (Activity Assay, Circular Dichroism, DSC, Native Page etc. to validate the refolding). Best, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll [pat.l...@drexel.edu] Sent: 20 June 2013 20:39 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type. Pat On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Kushol, No. The void for the column is 8ml and the whole volume of the column is 24ml. You must be talking about a different column. Zhen -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kushol Gupta Sent: Thursday, June 20, 2013 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Isn't 18 mLs into a Superdex 200 10/300 column run out near where the 670kD marker is, just after the void at ~15 mLs? Zhen, did you mean ~500kD rather than 5kD?. Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Laboratory Perelman School of Medicine University of Pennsylvania kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Hi Zhen, I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other techniques (Activity Assay, Circular Dichroism, DSC, Native Page etc. to validate the refolding). Best, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll [pat.l...@drexel.edu] Sent: 20 June 2013 20:39 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type. Pat On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.=
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Mea culpa - I'm thinking minutes at 0.5 ml/min, not mLs! (clearly I'm overdue for my afternoon caffeine...) Kushol -Original Message- From: Zhang, Zhen [mailto:zhen_zh...@dfci.harvard.edu] Sent: Thursday, June 20, 2013 4:18 PM To: 'Kushol Gupta'; CCP4BB@JISCMAIL.AC.UK Subject: RE: [ccp4bb] Puzzling observation about size exclusion chromatography Hi Kushol, No. The void for the column is 8ml and the whole volume of the column is 24ml. You must be talking about a different column. Zhen -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kushol Gupta Sent: Thursday, June 20, 2013 4:09 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Isn't 18 mLs into a Superdex 200 10/300 column run out near where the 670kD marker is, just after the void at ~15 mLs? Zhen, did you mean ~500kD rather than 5kD?. Kushol Kushol Gupta, Ph.D. Research Associate - Van Duyne Laboratory Perelman School of Medicine University of Pennsylvania kgu...@mail.med.upenn.edu 215-573-7260 / 267-259-0082 Hi Zhen, I'm not sure that binding to a monoclonal antibody is good evidence that the protein is in a natively folded state. I would be suspicious of such a result as the protein could be improperly, which is causing it to interact with the column matrix. It could be useful to use some other techniques (Activity Assay, Circular Dichroism, DSC, Native Page etc. to validate the refolding). Best, Rhys From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll [pat.l...@drexel.edu] Sent: 20 June 2013 20:39 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography If your protein elutes very late, that means it's binding to the column matrix (so all estimates of size go into the trash). Check to see that the ionic strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then the only solution is to go to a different matrix type. Pat On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail.=
Re: [ccp4bb] Puzzling observation about size exclusion chromatography
Hi Zhen - Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins. This is why the manufacturer recommends including a certain amount of salt in the running buffer. I have had the same experience with a few proteins, including one that came off the column well after the salt peak! (The protein was very clean after this step; all other proteins had eluted earlier.) As others have said, you can't rely on molecular weight calibrations in this case, but this behavior alone is no reason to think that the protein is misfolded or otherwise badly behaved. If you don't like the late elution, try increasing the salt concentration of your running buffer to 250 or even 500 mM. You'll probably need to exchange the eluted protein back into a low-salt buffer for your next steps (e.g. crystallization) if you do this. - Matt On 6/20/13 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. -- 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] Puzzling observation about size exclusion chromatography
Hi Matthew, Thanks a lot. That is a great idea. I will try the high salt and worry about the crystallization later. Zhen -Original Message- From: Matthew Franklin [mailto:mfrank...@nysbc.org] Sent: Thursday, June 20, 2013 4:34 PM To: Zhang, Zhen Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Puzzling observation about size exclusion chromatography Hi Zhen - Superdex is known to have some ion-exchange characteristics, so that it can weakly interact with some proteins. This is why the manufacturer recommends including a certain amount of salt in the running buffer. I have had the same experience with a few proteins, including one that came off the column well after the salt peak! (The protein was very clean after this step; all other proteins had eluted earlier.) As others have said, you can't rely on molecular weight calibrations in this case, but this behavior alone is no reason to think that the protein is misfolded or otherwise badly behaved. If you don't like the late elution, try increasing the salt concentration of your running buffer to 250 or even 500 mM. You'll probably need to exchange the eluted protein back into a low-salt buffer for your next steps (e.g. crystallization) if you do this. - Matt On 6/20/13 3:09 PM, Zhang, Zhen wrote: Dear all, I just observed a puzzling phenomenon when purifying a refolded protein with size exclusion chromatography. The protein was solubilized by 8M Urea and refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and is expected to be a trimer. The puzzling part is the protein after refolding always eluted at 18ml from the superdex S200 column (10/300), which is calculated to be 5KDal by standard. However, the fractions appear to be at 40KDal with SDS PAGE and the protein is functional in term of in vitro binding to the protein-specific monoclonal antibody. I could not explain the observation and I am wondering if anyone has the similar experience or has an opinion on this. Any comments are welcome. Thanks. Zhen The information in this e-mail is intended only for the person to whom it is addressed. If you believe this e-mail was sent to you in error and the e-mail contains patient information, please contact the Partners Compliance HelpLine at http://www.partners.org/complianceline . If the e-mail was sent to you in error but does not contain patient information, please contact the sender and properly dispose of the e-mail. -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
