Hi Paul, I used to use a large bore needle (~18-19 guage) and pass the sample through it a few time (more like 5-10 times); next use a small bore needle (~23 guage) to pass the sample another 5-10 times. Spin the sample at high speed (~10,000-12,000 rpm in a small Kubota or Eppendorf centrifuge - I'm not certain right now what this would be in terms of g, however), for a minute ; use the clear supernate for loading the gel. Since this is not quit an ultra centrifuge run, you will still have the membranes in the sample (so I always believe :))
If you are careful, the only sample loss you incur is the small amount that remain in the syringe and the needle - I used to try and get this out by pumping some air gently. Using a 1 mL syringe would also minimize losses. Hope that helps, Pow > > > > -----Original Message----- > > From: methods-boun...@oat.bio.indiana.edu [mailto: > > methods-boun...@oat.bio.indiana.edu] On Behalf Of Phelan, Paul J. > > Sent: Monday, April 27, 2015 2:45 PM > > To: meth...@magpie.bio.indiana.edu > > Subject: Whole cell extract gel samples for western blot > > > > Sometimes our gel samples (whole cell extracts) for western blots are > very > > viscous. What are the best methods for lysing small cell extracts well > > enough so that they don't float back out of the wells when loaded? > > > > Thank you, > > > > Paul Phelan > > Tufts University Boston > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > > > > _______________________________________________ > > Methods mailing list > > Methods@net.bio.net > > http://www.bio.net/biomail/listinfo/methods > > > _______________________________________________ > Methods mailing list > Methods@net.bio.net > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
