Re: [ccp4bb] BSA as additive
Dear H.Sin, These references may help you: Meireles, M., Aimar, P., and Sanchez, V. (2004) Albumin denaturation during ultrafiltration: effects of operating conditions and consequences on membrane fouling, Biotech. Bioeng. 38, 528-534. Schratter, P., (2004) Purification and concentration by ultrafiltration, Meth. Mol.Biol.: Protein Purification Protocols 244, 101-116 Sweryda-Krawiec, B., Devaraj, H., Jacob, G., and Hickman, J. J. (2004) A new interpretation of serum albumin surface passivation, Langmuir 20, 2054-2056. Adding 1% BSA to your buffer and pre-treating the centricon diafiltration membrane does prevent aggregation and improves protein yield by several fold. Additionally, adding 50 mM L-Arginine (pH’ed to buffer) prevented aggregation and produced nice protein crystals. Best wishes, Avinash
Re: [ccp4bb] BSA as additive
Regarding that paper, I would point out that cytosols generally contain 50-100 mM glutamate, so it makes sense that glutamate enhances solubility. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Evans, Nicola Sent: Monday, May 08, 2017 8:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] BSA as additive I haven't used BSA, but I did recently use 50mM L-glutamic acid for this exact reason (and 5% glycerol in all buffers except the last one for crystallography) after reading this paper and it made a big difference to my last protein prep: https://www.ncbi.nlm.nih.gov/pubmed/15264823 For my final crystallography buffer I have tried with and without L-glutamic acid (as I am trying to optimise micro-crystals and worried the L-glu would make sample too soluble) but still waiting to see if I get any improvement. Both have drops with micro-crystals already (after 2 days), the L-glutamic acid sample has fewer, hoping some other drops will yield better crystals over time. Hope that helps! Nicola From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Ha Sin <haab...@gmail.com<mailto:haab...@gmail.com>> Sent: 08 May 2017 12:32:44 To: CCP4BB@JISCMAIL.AC.UK<mailto:CCP4BB@JISCMAIL.AC.UK> Subject: [ccp4bb] BSA as additive Dear CCP4BB members, Sorry about my non crystallography question. Does anyone know of a reference where BSA has been used as an "additive" in the protein concentration step to prevent aggregation of the main protein? I would appreciate your suggestions. Thank you, H.Sin
Re: [ccp4bb] BSA as additive
I haven't used BSA, but I did recently use 50mM L-glutamic acid for this exact reason (and 5% glycerol in all buffers except the last one for crystallography) after reading this paper and it made a big difference to my last protein prep: https://www.ncbi.nlm.nih.gov/pubmed/15264823 For my final crystallography buffer I have tried with and without L-glutamic acid (as I am trying to optimise micro-crystals and worried the L-glu would make sample too soluble) but still waiting to see if I get any improvement. Both have drops with micro-crystals already (after 2 days), the L-glutamic acid sample has fewer, hoping some other drops will yield better crystals over time. Hope that helps! Nicola From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Ha Sin <haab...@gmail.com> Sent: 08 May 2017 12:32:44 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] BSA as additive Dear CCP4BB members, Sorry about my non crystallography question. Does anyone know of a reference where BSA has been used as an "additive" in the protein concentration step to prevent aggregation of the main protein? I would appreciate your suggestions. Thank you, H.Sin
Re: [ccp4bb] BSA as additive
(Sorry... over-enthusiastic sending) As for an actual reference - I'm not sure. It's a bit like lab folk-lore. Dave Dr David C Briggs Hohenester Lab Department of Life Sciences Imperial College London UK http://about.me/david_briggs From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Ha Sin <haab...@gmail.com> Sent: 08 May 2017 13:32:44 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] BSA as additive Dear CCP4BB members, Sorry about my non crystallography question. Does anyone know of a reference where BSA has been used as an "additive" in the protein concentration step to prevent aggregation of the main protein? I would appreciate your suggestions. Thank you, H.Sin
Re: [ccp4bb] BSA as additive
Hello, I've seen BSA used to "block" membranes of centrifugal concentrators when proteins were sticking to them. It appeared to work (proteins could be concentrated with less loss), but I'm not sure how much BSA ends up in your sample. I can imagine the answer is "a bit" and therefore for some techniques this might not be appropriate. Dave Dr David C Briggs Hohenester Lab Department of Life Sciences Imperial College London UK http://about.me/david_briggs From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Ha Sin <haab...@gmail.com> Sent: 08 May 2017 13:32:44 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] BSA as additive Dear CCP4BB members, Sorry about my non crystallography question. Does anyone know of a reference where BSA has been used as an "additive" in the protein concentration step to prevent aggregation of the main protein? I would appreciate your suggestions. Thank you, H.Sin
[ccp4bb] BSA as additive
Dear CCP4BB members, Sorry about my non crystallography question. Does anyone know of a reference where BSA has been used as an "additive" in the protein concentration step to prevent aggregation of the main protein? I would appreciate your suggestions. Thank you, H.Sin
