Dear Julius,

That is a good suggestion. I will definitely try this.

Thanks!


On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius <[log in to unmask]> 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 <[log in to unmask]> 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.