K+ and NH4+ ions precipitate SDS - forming the lovely cheese that makes it very difficult to load and run those samples.
Sometimes using LDS can help with the potassium issue. Heating the sample right before loading might help as well, or using a lot larger ratio of buffer to sample. Precipitation of protein prior to the gel is certainly an option, although some proteins have difficultues with recovering after that (membrane proteins in particular). Artem - Cosmic Cats approve of this message On Sat, Mar 2, 2019 at 9:53 PM Keller, Jacob <kell...@janelia.hhmi.org> wrote: > 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> 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 > LinkedIn: 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
