I have tagged my 12kDa eIF1 protein with Fluorescein-maleimide (Ex: 485nm, Em: 530nm). The interaction with this particular protein with 40S ribosome should show anisotropy value change. 10nM of eIF1_FL and 40nM of 40S in proper binding buffer (25mM HEPES-KOH pH 7.5, 2.5mM Mg(OAc)2, 80mM KOAc, 2mM DTT) were incubated and vertical and horizontal polarization measured. Anisotropy values calculated (Taking G=1) from those polarization values shows negative digits:
Blank Buffer: 0.123
Only eIF1_FL (10nM): -0.295
eIF1_FL (10nM) + 40S (40nM): -0.302
eIF1_FL (10nM) + 40S (200nM): -0.301
Please suggest how to improve my reaction conditions to get a values in the range of expected one.
Because you did not apply a G-factor correction, your anisotropy measurements are relative. A negative value means that the perpendicular fluorescence intensity was higher than the parallel intensity.
Negative anisotropy is a possibility. The values can be between -0.2 and +0.4. However, in your case, it is probably due to the fact the the detection sensitivity of the perpendicular measurement is higher than the detection sensitivity of the parallel measurement. This is due to the instrument, not the sample. That is why a G-factor correction is used. In order to measure the G-factor, you require both horizontal and vertical excitation polarizer positions.
See this article for the definition of the G-factor and how it is applied.
https://en.wikipedia.org/wiki/Fluorescence_anisotropy
Thanks a lot Adam. It was a wrong feed of equation in the instrument itself. And a correction of G-factor is giving the desired reading.
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