Hi Jacob, If you keep the protein solution warm the KDS remains soluble. Just load the sample on the gel immediately after heating to denature. This has allowed me to load samples containing up to 1.5M KCl with no problems.
Best wishes, Steve Dr Stephen Carr Research Complex at Harwell (RCaH) Rutherford Appleton Laboratory Harwell Oxford Didcot Oxon OX11 0FA United Kingdom Email stephen.c...@rc-harwell.ac.uk tel 01235 567717 ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Bonsor, Daniel <dbon...@som.umaryland.edu> Sent: 03 March 2019 03:19:34 To: ccp4bb Subject: Re: [ccp4bb] KCl in SDS-PAGE workarounds? SDS is soluble, whereas the potassium salt of dodecyl sulphate is insoluble. You could try 18-Crown-6 ether to chelate the potassium, though I don't know if the crown ether would then affect the gel. Dan Daniel A. Bonsor PhD Institute of Human Virology, University of Maryland at Baltimore 725 W. Lombard Street N571 Baltimore MD 21201 ________________________________ From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Keller, Jacob <kell...@janelia.hhmi.org> Sent: Saturday, March 2, 2019 9:52 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] KCl in SDS-PAGE workarounds? Dear crystallographers, It has been my experience that KCl does nasty things when loading SDS-PAGE gels. Does anyone have an easy workaround, perhaps TCA precipitation? Ideally this would be something nicely quantitative yet quick and easy…. Any suggestions appreciated. All the best, Jacob Keller +++++++++++++++++++++++++++++++++++++++++++++++++ Jacob Pearson Keller Research Scientist / Looger Lab HHMI Janelia Research Campus 19700 Helix Dr, Ashburn, VA 20147 Desk: (571)209-4000 x3159 Cell: (301)592-7004 +++++++++++++++++++++++++++++++++++++++++++++++++ The content of this email is confidential and intended for the recipient specified in message only. It is strictly forbidden to share any part of this message with any third party, without a written consent of the sender. If you received this message by mistake, please reply to this message and follow with its deletion, so that we can ensure such a mistake does not occur in the future. From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Artem Evdokimov Sent: Saturday, March 2, 2019 8:32 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins Hi Alex, In my experience you just have to experiment with these parameters (in however of a limited space that is afforded by your experimental set-up). If you have the right tools you can slog through semifactorial or DoE-based scouting of various parameters with relative ease. These parameters typically include pH, salinity, detergent(s), and other variables (e.g. the nature of the buffer and salt(s) - many cases exist where the 'standard' choices do not work well). Literature-based analogies do help on occasion. For example, membrane-spanning p450-type proteins often prefer high salt, and often would do better in sodium acetate as opposed to chloride. I've worked with ion channels that preferred 3M NaCl, or 2M MgCl2 and other fairly weird conditions... Artem - Cosmic Cats approve of this message On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín <aperalva...@gmail.com<mailto:aperalva...@gmail.com>> wrote: Dear all, any reference as a guide for selecting the appropriate salt and concentration for membrane proteins extraction buffer? Best, Alex -- Alex Perálvarez-Marín, Ph.D. Centre d'Estudis en Biofísica / Unitat de Biofísica Edifici M Universitat Autònoma de Barcelona 08193 Cerdanyola del Vallés Barcelona Spain Phone: +34 93 581 4504 FAX: +34 93 581 1907 e-mail: aperalva...@gmail.com<mailto:aperalva...@gmail.com> LinkedIn: es.linkedin.com/in/aperalvarez/<http://es.linkedin.com/in/aperalvarez/> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 This email and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. 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