Dear Julius, That is a good suggestion. I will definitely try this.
Thanks! On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius <[email protected]> wrote: > Dear Mohammad, > your buffer is key to success in biophysical measurements. While your > protein may be totally fine in standard buffer, the large and hydrophobic > fluorophores used in FP, MST and other assays are prone to adsorbing to > plasticware etc. Also, at 1nM concentration, you are losing much more of > your protein to adsorption, so you will have far less active protein at > those concentrations than calculated. I would try to add detergent and, if > necessary, BSA to your buffer. For my assay development projects, Tween20 > or Brij35 (0.03%) have worked well and I have used 0.5% BSA. Once you get > the buffer right, measurements will be far more reproducible. Also, try to > include a good control, e.g. DNA with Cy3, but sequence that does not bind > to test for nonspecific, concentration dependent effects. > I hope this helps, if you have any questions, feel free to email me! > Best, > Julius > > > > On 21 Jul 2017, at 15:32, Mohammad Khan <[email protected]> wrote: > > > > 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. > >
