Re: [ccp4bb] off-topic: bug busting
Dear Phoebe, We have a constant systems homogenizer that we use routinely as a service for researchers here at UGA. It is really easy to use and gets up to high pressure (40k psi) so you can lyse plant cells or other difficult to lyse cell types. You just pour/pump your resuspended cells, as low as 25mL, into the inlet, setup parameters (usually once) and hit the start button. The lysed cells will exit the outlet line. We usually have 2 cups to catch the outlet and we recycle 3-6 times. The unit will automatically shutoff when it runs out of liquid. Below is a link to the website for the company with a video. For greater than 1L of cells we have a Niro-Soavi high pressure homogenizer (second link) that I recommend. http://www.constantsystems.com/products/cell%20disruption%20systems/ts%20series%20cabinet http://www.nirosoavi.com/products/Ariete_NS2006.asp On Tue, Feb 4, 2014 at 11:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
Interesting 'off-topic' thread. I'm a rather long-time user of Microfluidics cell disruptors (for E. coli or P. pastoris or S. ceresvisiae), and a shared-use M110-P Plug and Play is used by most of the membrane-heads at my place. I've generally been happy (or at least not unhappy) with it. However, we've been having some QC issues with a membrane protein that we're making in S. ceresvisiae (Sc), and I'm having some concerns about sample heating. Can anyone comment on Microfludics vs Avestin vs Constant Systems vs Retsch vs whatever-else for cracking Sc cells? These days, we're working up ~300-400g of paste at a time. Thank you very much! -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Wed, 5 Feb 2014 00:34:11 -0500 Anirban Banerjee ani...@gmail.com wrote: I will be curious to know about people's experiences with membrane proteins and lysing yeast cells with the Microfluidizer and how that compares with using a Retsch Miller, i.e. grinding in a liquid nitrogen cooled stainless steel chamber and plunging in liquid nitrogen in between grinding cycles. I am worried that the Microfluidizer is not as mild w.r.t. heating as they claim it to be. That would, of course, perfectly qualify as my OCD. Any insights will be really appreciated. Thanks, Anirban On Tue, Feb 4, 2014 at 11:58 AM, Matthew Franklin mfrank...@nysbc.org wrote: Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- 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] off-topic: bug busting
If heat is a concern, you can place your Emulsiflex into a bin with lots of ice. We have the version with cooling after lysis, but if you are paranoid you can cool the whole thing. It has the footprint of a 15” MacBook Pro but it’s way more expensive than that :-) Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Feb 5, 2014, at 11:49 AM, Michael C. Wiener mwie...@virginia.edumailto:mwie...@virginia.edu wrote: Interesting 'off-topic' thread. I'm a rather long-time user of Microfluidics cell disruptors (for E. coli or P. pastoris or S. ceresvisiae), and a shared-use M110-P Plug and Play is used by most of the membrane-heads at my place. I've generally been happy (or at least not unhappy) with it. However, we've been having some QC issues with a membrane protein that we're making in S. ceresvisiae (Sc), and I'm having some concerns about sample heating. Can anyone comment on Microfludics vs Avestin vs Constant Systems vs Retsch vs whatever-else for cracking Sc cells? These days, we're working up ~300-400g of paste at a time. Thank you very much! -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Wed, 5 Feb 2014 00:34:11 -0500 Anirban Banerjee ani...@gmail.commailto:ani...@gmail.com wrote: I will be curious to know about people's experiences with membrane proteins and lysing yeast cells with the Microfluidizer and how that compares with using a Retsch Miller, i.e. grinding in a liquid nitrogen cooled stainless steel chamber and plunging in liquid nitrogen in between grinding cycles. I am worried that the Microfluidizer is not as mild w.r.t. heating as they claim it to be. That would, of course, perfectly qualify as my OCD. Any insights will be really appreciated. Thanks, Anirban On Tue, Feb 4, 2014 at 11:58 AM, Matthew Franklin mfrank...@nysbc.orgmailto:mfrank...@nysbc.org wrote: Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- 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] off-topic: bug busting
We always have used ice with the Microfluidics disruptor. We also, on occasion, monitor the temperature of the process stream. I'm not convinced, at all, that there is actually any problem with heating, but I thought it reasonable to put the query out there. Thanks to all who emailed me. -MW On Wed, 5 Feb 2014 12:07:18 -0500 Bosch, Juergen jubo...@jhsph.edu wrote: If heat is a concern, you can place your Emulsiflex into a bin with lots of ice. We have the version with cooling after lysis, but if you are paranoid you can cool the whole thing. It has the footprint of a 15” MacBook Pro but it’s way more expensive than that :-) Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Feb 5, 2014, at 11:49 AM, Michael C. Wiener mwie...@virginia.edumailto:mwie...@virginia.edu wrote: Interesting 'off-topic' thread. I'm a rather long-time user of Microfluidics cell disruptors (for E. coli or P. pastoris or S. ceresvisiae), and a shared-use M110-P Plug and Play is used by most of the membrane-heads at my place. I've generally been happy (or at least not unhappy) with it. However, we've been having some QC issues with a membrane protein that we're making in S. ceresvisiae (Sc), and I'm having some concerns about sample heating. Can anyone comment on Microfludics vs Avestin vs Constant Systems vs Retsch vs whatever-else for cracking Sc cells? These days, we're working up ~300-400g of paste at a time. Thank you very much! -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Wed, 5 Feb 2014 00:34:11 -0500 Anirban Banerjee ani...@gmail.commailto:ani...@gmail.com wrote: I will be curious to know about people's experiences with membrane proteins and lysing yeast cells with the Microfluidizer and how that compares with using a Retsch Miller, i.e. grinding in a liquid nitrogen cooled stainless steel chamber and plunging in liquid nitrogen in between grinding cycles. I am worried that the Microfluidizer is not as mild w.r.t. heating as they claim it to be. That would, of course, perfectly qualify as my OCD. Any insights will be really appreciated. Thanks, Anirban On Tue, Feb 4, 2014 at 11:58 AM, Matthew Franklin mfrank...@nysbc.orgmailto:mfrank...@nysbc.org wrote: Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- 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] off-topic: bug busting
Dear all, Our lab is predominantly interested in membrane proteins and currently uses a Constant Systems device for E coli hooked up to a cooling unit which pumps 4C water around the outside of the main chamber and keeps the sample cool even when using large volumes. This works really well for E. coli but we are expressing/studying more and more (mycobacterial) proteins in M. smegmatis these days and have found that our French press is still far superior to the Constant Systems device. The French press is aging though and I was interested if anyone else has experience with lysing M. smeg and if so which system/device they would recommend? Cheers Adam Dr Adam Heikal Department of Microbiology and Immunology University of Otago +64 (3) 471-6464 adam.hei...@otago.ac.nz http://micro.otago.ac.nz/our-people/adamheikal On 6/02/14 5:49 AM, Michael C. Wiener mwie...@virginia.edu wrote: Interesting 'off-topic' thread. I'm a rather long-time user of Microfluidics cell disruptors (for E. coli or P. pastoris or S. ceresvisiae), and a shared-use M110-P Plug and Play is used by most of the membrane-heads at my place. I've generally been happy (or at least not unhappy) with it. However, we've been having some QC issues with a membrane protein that we're making in S. ceresvisiae (Sc), and I'm having some concerns about sample heating. Can anyone comment on Microfludics vs Avestin vs Constant Systems vs Retsch vs whatever-else for cracking Sc cells? These days, we're working up ~300-400g of paste at a time. Thank you very much! -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Wed, 5 Feb 2014 00:34:11 -0500 Anirban Banerjee ani...@gmail.com wrote: I will be curious to know about people's experiences with membrane proteins and lysing yeast cells with the Microfluidizer and how that compares with using a Retsch Miller, i.e. grinding in a liquid nitrogen cooled stainless steel chamber and plunging in liquid nitrogen in between grinding cycles. I am worried that the Microfluidizer is not as mild w.r.t. heating as they claim it to be. That would, of course, perfectly qualify as my OCD. Any insights will be really appreciated. Thanks, Anirban On Tue, Feb 4, 2014 at 11:58 AM, Matthew Franklin mfrank...@nysbc.org wrote: Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=artic leid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
[ccp4bb] off-topic: bug busting
Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu mailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- 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] off-topic: bug busting
Emulsiflex C5 alternative freeze-thawing, the old way we used to do this spiking with some Lysozyme or grinding it in a LN2 cooled mortar, is very effective and pretty cost effective, assuming you don’t pay for the LN2 Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-2926tel:%2B1-410-955-2926 http://lupo.jhsph.edu On Feb 4, 2014, at 11:49 AM, Phoebe A. Rice pr...@uchicago.edumailto:pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edumailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
Hi Phoebe, Another possibility is the Emulsiflex (from Avestin, in canada). Not a cheap piece of equipment, but very sturdy and efficient to up to 200 mL of extract (runs on house-air). Can deal with E coli (and even yeast if you have the models with internal compressor). It comes in 3 sizes I think we have the middle one (C-3) with a compressor. It is basically a french press but without the inconvenient of being french (I am french myself). It is quite gentle, and does not overheat samples as much as the sonicator. We make a lot of membrane protein purifications and I have been working with this since my post-doc Hope this helps. Best regards, Pascal Egea On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] off-topic: bug busting
LOL. In any case, a French press is also not french, according to Wikipedia it was invented by Charles Stacy French of the Carnegie Institution of Washington (hence French press with capital F). In any case, I agree, Emulsiflex, Constant Systems One-shot, Microfluidizer etc. is the way to go if you have some money. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 4 Feb 2014, at 18:09, Pascal Egea wrote: Hi Phoebe, Another possibility is the Emulsiflex (from Avestin, in canada). Not a cheap piece of equipment, but very sturdy and efficient to up to 200 mL of extract (runs on house-air). Can deal with E coli (and even yeast if you have the models with internal compressor). It comes in 3 sizes I think we have the middle one (C-3) with a compressor. It is basically a french press but without the inconvenient of being french (I am french myself). It is quite gentle, and does not overheat samples as much as the sonicator. We make a lot of membrane protein purifications and I have been working with this since my post-doc Hope this helps. Best regards, Pascal Egea On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] off-topic: bug busting
Adapted from periplasmic fractionation protocol, but for cytoplasmic proteins: 1. Incubate resuspended cells with lysozyme in normal buffer but with 20% sucrose. 2. Spin down sphaeroplasts (unbroken cells with no cell wall - ultra fragile). This also removes periplasmic proteases. 3. Resuspend white pellet in buffer of choice and freeze-thaw. Or dilute buffer for osmotic shock. Or sonicate *very* lightly - perhaps using an aging sonicator! 4. Gives complete lysis from 2 ml to 2 litre culture volumes very reproducibly, and avoids proprietary bug-buster type detergent mixes. See this paper - it also greatly increases purification (10-fold) of low abundance his tag proteins that are otherwise outcompeted by periplasmic components (siderophores?) http://www.nature.com/nmeth/journal/v6/n7/full/nmeth0709-477.html Darren On 04/02/14 17:49, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu mailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- ** Dr. Darren J. Hart, CNRS Research Director, Unit of Virus Host Cell Interactions (UVHCI) Unité Mixte Internationale UMI 3265 (CNRS-EMBL-UJF) Director, Integrated Structural Biology Grenoble (ISBG) Unité Mixte de Service UMS 3518 (CNRS-CEA-UJF-EMBL) ** Email: h...@embl.fr Tel: +33 4 76 20 77 68; Fax: +33 4 76 20 71 99 Postal address: UVHCI/ISBG, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France For funded access to ESPRIT construct screening via EU FP7 BioStruct-X: www.biostruct-x.eu/ **
Re: [ccp4bb] off-topic: bug busting
I would also recommend the Emulsiflex line of cell disruptors from Avestin - it has been my favorite and most easily used instrument of cell destruction. On Tue, Feb 4, 2014 at 9:17 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote: LOL. In any case, a French press is also not french, according to Wikipedia it was invented by Charles Stacy French of the Carnegie Institution of Washington (hence French press with capital F). In any case, I agree, Emulsiflex, Constant Systems One-shot, Microfluidizer etc. is the way to go if you have some money. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 4 Feb 2014, at 18:09, Pascal Egea wrote: Hi Phoebe, Another possibility is the Emulsiflex (from Avestin, in canada). Not a cheap piece of equipment, but very sturdy and efficient to up to 200 mL of extract (runs on house-air). Can deal with E coli (and even yeast if you have the models with internal compressor). It comes in 3 sizes I think we have the middle one (C-3) with a compressor. It is basically a french press but without the inconvenient of being french (I am french myself). It is quite gentle, and does not overheat samples as much as the sonicator. We make a lot of membrane protein purifications and I have been working with this since my post-doc Hope this helps. Best regards, Pascal Egea On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu -- Jim Fairman, Ph D. Crystal Core Leader I Emerald Bio http://www.embios.com Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] off-topic: bug busting
BeadBeater. http://biospec.com/. Gentle, aerosol-free way to break 15-350 mL of cell paste (2-150 g wet packed cells). Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/4/2014 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu mailto:pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
3 to 1 ratio of bper and yper, 2-3 ml per gram of e. coli plus lysozyme and bensonaze... I have not used a sonicator in years :) about 1% of proteins dont like detergents, in which case there are other non mechanical options. A. On Feb 4, 2014 10:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
A lot of ageing sonicators are not really ageing. Have you tried cleaning the tip of the sonicator? If not, remove the tip from the sonicator and use a 800 Grit wet sandpaper to make it as shiny as it was when you first bought it. I do this every three times I use the sonicator. A lot of crap deposits on it with every use. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 Original message Date: Tue, 4 Feb 2014 12:40:51 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Roger Rowlett rrowl...@colgate.edu) Subject: Re: [ccp4bb] off-topic: bug busting To: CCP4BB@JISCMAIL.AC.UK BeadBeater. http://biospec.com/. Gentle, aerosol-free way to break 15-350 mL of cell paste (2-150 g wet packed cells). Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/4/2014 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
I'm joining the pig-pile on the Emulsiflex. I like it because: a. The folks at Avestin are very helpful (they're Canadian--just naturally nice) b. It'll break yeast as well as E coli c. It seems pretty gentle to me (we pack it in ice, so there's negligible sample heating) d. It's reproducible--if I put a sample through twice, I KNOW it'll be well broken, whereas I'm never really sure with a sonicator (maybe the tip is aging/dirty/pitted, maybe it's not tuned properly, etc. etc...of course this may just be my OCD talking) e. It gave me an excuse to go to Sears and buy a big hunkin' air compressor Having said that, it requires regular lubrication, and periodic replacement of valves, etc., but the vendor is very helpful about talking you through maintenance (and they have at least one person who travels a lot, who will stop by while he's in town, should we need hand-holding). Pat On 4 Feb 2014, at 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
[ccp4bb] Stacey French (was Re: [ccp4bb] off-topic: bug busting)
Govindjee and Fork's biography of Stacey French is available here: http://www.nasonline.org/publications/biographical-memoirs/memoir-pdfs/french-c-stacy.pdf or if your library subscribes to photosynthesis research, http://link.springer.com/article/10.1007%2Fs11099-006-0041-6 Mark J van Raaij wrote: LOL. In any case, a French press is also not french, according to Wikipedia it was invented by Charles Stacy French of the Carnegie Institution of Washington (hence French press with capital F). In any case, I agree, Emulsiflex, Constant Systems One-shot, Microfluidizer etc. is the way to go if you have some money. Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 4 Feb 2014, at 18:09, Pascal Egea wrote: Hi Phoebe, Another possibility is the Emulsiflex (from Avestin, in canada). Not a cheap piece of equipment, but very sturdy and efficient to up to 200 mL of extract (runs on house-air). Can deal with E coli (and even yeast if you have the models with internal compressor). It comes in 3 sizes I think we have the middle one (C-3) with a compressor. It is basically a french press but without the inconvenient of being french (I am french myself). It is quite gentle, and does not overheat samples as much as the sonicator. We make a lot of membrane protein purifications and I have been working with this since my post-doc Hope this helps. Best regards, Pascal Egea On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] off-topic: bug busting
Or if the tip is hopelessly pitted you can get a new one (for a Branson 1/2 inch horn) at: http://www.proequip.com/productlist.asp?pcid=31 (thanks to: http://blogs.cornell.edu/collinslab/2010/04/12/time-for-a-new-tip/ ) Reza Khayat wrote: A lot of ageing sonicators are not really ageing. Have you tried cleaning the tip of the sonicator? If not, remove the tip from the sonicator and use a 800 Grit wet sandpaper to make it as shiny as it was when you first bought it. I do this every three times I use the sonicator. A lot of crap deposits on it with every use. Best wishes, Reza Reza Khayat, PhD Assistant Professor The City College of New York Department of Chemistry, MR-1135 160 Convent Avenue New York, NY 10031 Tel. (212) 650-6070 Original message Date: Tue, 4 Feb 2014 12:40:51 -0500 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Roger Rowlett rrowl...@colgate.edu) Subject: Re: [ccp4bb] off-topic: bug busting To: CCP4BB@JISCMAIL.AC.UK BeadBeater. http://biospec.com/. Gentle, aerosol-free way to break 15-350 mL of cell paste (2-150 g wet packed cells). Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/4/2014 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp
Re: [ccp4bb] off-topic: bug busting
I will be curious to know about people's experiences with membrane proteins and lysing yeast cells with the Microfluidizer and how that compares with using a Retsch Miller, i.e. grinding in a liquid nitrogen cooled stainless steel chamber and plunging in liquid nitrogen in between grinding cycles. I am worried that the Microfluidizer is not as mild w.r.t. heating as they claim it to be. That would, of course, perfectly qualify as my OCD. Any insights will be really appreciated. Thanks, Anirban On Tue, Feb 4, 2014 at 11:58 AM, Matthew Franklin mfrank...@nysbc.org wrote: Hi Phoebe - Cost-effective may not be the applicable word here, but the Microfluidizer works very well: http://www.microfluidicscorp.com/index.php?option=com_contentview=articleid=19Itemid=76 This gadget runs on house compressed air (don't try to use a compressed air tank - you'll empty it in minutes). It's a bit noisy, but so is a sonicator. The Microfluidizer really shines with large volumes of lysate - like 1 L and up. If you're only processing 100-200 mL at a time, I think sonication is the best way to go. Hope that helps, Matt On 2/4/14 11:49 AM, Phoebe A. Rice wrote: Some time ago, there was a nice discussion of cost-effective, wimpy protein-friendly ways to break open E. coli. We're thinking about replacing an aging sonicator. If people have a favorite gizmo, could they repeat that advice? thank you, Phoebe Rice ++ Phoebe A. Rice Dept. of Biochemistry Molecular Biology The University of Chicago 773 834 1723; pr...@uchicago.edu http://bmb.bsd.uchicago.edu/Faculty_and_Research/ http://www.rsc.org/shop/books/2008/9780854042722.asp -- Matthew Franklin, Ph. D. Senior Scientist New York Structural Biology Center 89 Convent Avenue, New York, NY 10027 (212) 939-0660 ext. 9374
