[ccp4bb] Detergent solubilization step (membrane proteins)
Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Raji, First, I would suggest increasing the solubilization time by attempting solubilization overnight at 4 degrees. I would also suggest a few more detergent choices. In addition to DDM, my other favories are OG and Elugent (a mixture of several detergents) for extraction from *E. coli* membranes. If one of these still doesn't extract your protein of interest, you can move onto other more exotic options. I know it's a bit self-plugging, but the products division of Emerald Bio sells a 96-well detergent screen you can use to go through a wide variety of solubilization detergent options at small scale. We've used it in-house in our services division to get some nice results for several of our membrane protein projects. However, you may be fighting a losing battle as many times protein that doesn't extract from the membranes can be misfolded or aggregated, remaining strongly associated with the membranes. Cheers, Jim On Wed, Jul 10, 2013 at 2:23 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Hi Raji, the amount of detergent used is more important than the concentration. The more cells you have, the more detergent is required. To give you an idea, we tend to use 100 ml of 1% detergent (=1 g) for 12 liters of cells (Ecoli) of OD600 1. This is probably on the low side; if cost weren't an issue I'd prefer using at least 2% (2 g) for this amount of cells. So, the lower extraction yield for your second protein could simply be due to higher ODs. To increase yields you can either (as Jim said) increase extraction time and/or increase the amount of detergent. Also, the milder the detergent (like DDM), the less efficient it tends to be in extraction. So, 1 g of DM will be more efficient than 1 g of DDM, but of course the DM may not keep your protein happy.For very stable proteins, the detergents to use are LDAO and Elugent (a mixture of alkylglucosides); these are also very cheap compared to DDM. A final possibility is indeed that some of the protein may be misfolded and unsolubilizable. Good luck, Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Wednesday, July 10, 2013 10:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Detergent solubilization step (membrane proteins) Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
I could add that even for membrane proteins that may not be stable in eg DM after purification, DM may still be a viable option for extraction since there will be large amounts of stabilising lipids present. Just make sure you change to a milder detergent for the post-extraction steps. Bert From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Raji Edayathumangalam [r...@brandeis.edu] Sent: Wednesday, July 10, 2013 10:23 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Detergent solubilization step (membrane proteins) Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] Detergent solubilization step (membrane proteins)
Thanks Jim and Bert for all your suggestions. I plan to increase time and/or amount of DDM and see what happens before switching detergents. I'll keep the suggestions about other detergents and 96-detergent screen in the back of my mind. Bert, I noted your suggestion of also using a larger amount of total detergent and will definitely try that. Jim, thanks for suggesting Elugent. Never heard about it before so great to know. Many thanks and cheers, Raji On Wed, Jul 10, 2013 at 5:23 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Dear BBers, Sorry for the non-ccp4 post. I'd like to hear tips and suggestions from the membrane protein folks in the community. I am currently purifying two different membrane proteins expressed in E. coli. While I am able to extract practically all of my first protein from the cell membrane into buffer containing 1% DDM in 1hr at 4C, it doesn't seem as though that works very well for my second protein. I understand every protein is a unique beast; I am just trying to increase the yields for my second protein as much as possible. For my second protein, I have tried solubilizing for 1hr at 4C in 1% DDM as well as in 1-2% NM for 1hr at 4C but am only able to solubilize about half of total protein in the membrane. Have folks seen substantial increase in % solubility with longer incubations with detergent? Or should I consider the issue that the fraction that doesn't solubilize may be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
