I am doing nucleotide exchange assay using Tecan spark 10 M machine.I have loaded KRas with MANT-GDP and used 1uM of the same and the relative Fluorescence units is around 32000. But the nucleotide exchange buffer ( 20 mM HEPES, 150 mM NaCl pH7.5) is also showing similar fluorescence value. I added SOS (GEF) and 20 fold molar excess GTP but the fluorescence value is not decreasing. So anyone encountered this before? Why my Nucleotide exchange not working? Excitation wavelength used is 360 nm and emission-450 nm.
Based on your statement that the fluorescence intensity was the same for 1 µM MANT-GDP and the buffer, it seems that your instrument is not detecting the MANT-GDP. The wavelength settings are correct for MANT-GDP.
What type of plate are you using? My recommendation is to use a black plate with top reading. Prepare serial dilutions of MANT-GDP in the buffer. Include 0.01% Triton-X100 or Tween-20 in the buffer to prevent things from sticking to the plastic. Optimize the gain and focal height settings using the highest MANT-GDP concentration to start with. 1 µM should be high enough, but if not, try 10 µM.
Hi Adam,
Yes I am using black plate with top reading. I will add Triton X-100 and see whether I can get low RFU value for buffer only or not. Gain fluorescence used was 75% of maximum. have tried adding SOS protein which is supposed to decrease fluorescence value due to nucleotide exchange but that is not happening. Thanks for suggestion. I will repeat and see if it works out.
I have used the method suggested by Sebastian Schmitt
. It works for tightly bound nucleotides (nM Kd), but weakly bound ones (µM Kd) are removed by the gel filtration. You can use smaller gel filtration spin columns than PD-10s if you have limited protein to work with.After denaturing the protein with perchloric acid, the perchloric acid can be precipitated with 1/2 equivalent of potassium carbonate, leaving the nucleotides in the supernatant at neutral pH.
Thanks for your answer. Adam B Shapiro you rightly pointed out my instrument was not detecting MANT-GDP. I did the optimisation of MANT-GDP and added triton X-100 to the buffer to prevent things for sticking to the plastic. MANT-GDP is not tightly bound so I will change the buffer and use PD-10.
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