Dear Thomas, I do all my experiments in a 384 well, non-binding plates. I have a salt concentration of 50 mM NaCl, with BSA. i will try the other suggestions as well.
>From a couple of experiments, I could determine the Kd to be approx. 10 nM. Thanks for all the suggestions! On Fri, Jul 21, 2017 at 4:11 PM, Thomas Edwards <t.a.edwa...@leeds.ac.uk> wrote: > A few tips, some/all of which may be relevant. Or not… > > > > If you are in 96 or 384 well plates, they vary. We tried quite a few > batches and brands before settling on one. > > > > Keep your salt concentration as low as possible. > > > > You should be able to measure Kd in a range of about 1nM to low uM. > Outside that range is harder, or possibly impossible. > > > > Buffers matter. > > RNA binding proteins have preferred phosphates, PPIs Tris. > > Some triton and/or some BSA can help. > > > > All proteins are different, and each likes some or all or none of the > above….. > > > > Ideally you should convert polarization to anisotropy. Simple enough – but > some referees can get picky… > > > > *Ed* > > > > *T.A.Edwards Ph.D.* > > *Deputy Director* Astbury Centre for Structural Molecular Biology > > Ass. Professor, School of Molecular and Cellular Biology > > Garstang 8.53d > > University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 > > http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE > > Invention, my dear friends, is 93% perspiration, 6% electricity, 4% > evaporation, and 2% butterscotch ripple. ~Willy Wonka > > > > *From: *CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Mohammad > Khan <mohdkhan0...@gmail.com> > *Reply-To: *Mohammad Khan <mohdkhan0...@gmail.com> > *Date: *Friday, 21 July 2017 at 14:32 > *To: *"CCP4BB@JISCMAIL.AC.UK" <CCP4BB@JISCMAIL.AC.UK> > *Subject: *[ccp4bb] Off topic: Flourescence anisotropy measurement > > > > Dear all, > > > > I am trying to measure the difference in polarization upon the binding of > the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying > dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in > difference of polarization with decrease in protein concentration. However, > the results are difficult to reproduce and also vary greatly within > triplicates of an experiment. > > > > Similar observations have been observed by my colleagues with their > proteins. > > > > Are there any tips or precautions to keep in mind while setting up these > reactions? > > > > Looking forward for suggestions. > > > > Thank you. >
