Re: [ccp4bb] Ion exchange protein lost
Like Juergen suggested, your protein is most likely stuck to the column, either because it never was really was refolded or because it doesn't like to be at pH 4.5. Do you really need such a low pH to have it bind to source 15S. Check the pI of the protein and perhaps some different pH buffer or try an anion exchange column if possible. Ursula On 5/11/10 7:53 PM, megha goyal wrote: Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg -- Ursula Schulze-Gahmen, PhD. QB3, Tjian Lab MCB, 16 Barker Hall #3204 University of California Berkeley Berkeley, CA 94720-3204 Phone: (510) 642 8258 uschulze-gah...@lbl.gov
Re: [ccp4bb] Ion exchange protein lost
Human gcsf has a pI ~ 6. As Ursula suggests you might be better off with anion exchange. There are protocols that show you can both refold and purify onto such column type. Nadir Pr. Nadir T. Mrabet Structural Molecular Biochemistry Nutrigenex - INSERM U-954 Nancy University, School of Medicine 9, Avenue de la Foret de Haye, BP 184 54505 Vandoeuvre-les-Nancy Cedex France Phone: +33 (0)3.83.68.32.73 Fax: +33 (0)3.83.68.32.79 E-mail: Nadir.Mrabetat medecine.uhp-nancy.fr Cell.: +33 (0)6.11.35.69.09 LEGAL NOTICE Unless expressly stated otherwise, this message is confidential and may be privileged. It is intended for the addressee(s) only. Access to this E-mail by anyone else is unauthorized. If you are not an addressee, any disclosure or copying of the contents of this E-mail, or any action taken (or not taken) in reliance on it, is unauthorized and may be unlawful. If you are not an addressee, please inform the sender immediately. On 12/05/2010 19:17, Ursula Schulze-Gahmen wrote: Like Juergen suggested, your protein is most likely stuck to the column, either because it never was really was refolded or because it doesn't like to be at pH 4.5. Do you really need such a low pH to have it bind to source 15S. Check the pI of the protein and perhaps some different pH buffer or try an anion exchange column if possible. Ursula On 5/11/10 7:53 PM, megha goyal wrote: Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg -- Ursula Schulze-Gahmen, PhD. QB3, Tjian Lab MCB, 16 Barker Hall #3204 University of California Berkeley Berkeley, CA 94720-3204 Phone: (510) 642 8258 uschulze-gah...@lbl.gov
[ccp4bb] Ion exchange protein lost
Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg
Re: [ccp4bb] Ion exchange protein lost
Hi Megha, how about your protein is stuck to the top of your column ? How does your pressure look like before injection and after injection ? What makes you believe that you have successfully refolded your protein ? Just some thoughts, Jürgen On May 11, 2010, at 10:53 PM, megha goyal wrote: Hi all, Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration using proflux M12 [just concentration and not diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S resin [strong cation exchange]. The problem is we do not recover our protein on performing IEX. HPLC and absorbance reading on concentrate show the presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and even 2M Nacl washing does not show our protein. . Only when we perform NaOH wash we do see some peak but could not analyse it as it is too alkaline and cant run on SDS PAGE or HPLC. What could be the reason. Where do we lose our protein. Kindly shed some light on this on where shall I be going wrong. thanks and regards, meg - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
