My protein shows intrinsic tryptophan flourescence. I used this property to study the lifetime properties. In the same context i performed acrylamide and KI based quenching methods to study accessibility of the probe in presence and absence of an inhibitor. In the protein without inhibitor I see gradual quenching of fluorescence as expected however, on addition of the inhibitor I see an erratic pattern. There is no consistent decrease in fluorescence intensity. I changed the quencher from acrylamide to potassium iodide (sodium thiosulphate) but it has not helped. I have to calculate the kq value. Please give suggestions to solve the issue. * protein samples were filtered after addition of inhibitor to remove any aggregates if formed.
It would help to know more about this "erratic pattern." Is the result consistent but just not monotonic? Perhaps there is an interaction between the inhibitor and the quencher, or there could be a solubility issue with the inhibitor. Poor inhibitor solubility could be made even worse by the high ionic strength of the quencher.
Hi Adam. Thank you for your answer. To add on to query, I see an increase in fluorescence intensity on addition of the quencher in the initial concentrations followed by random increase or decrease with higher concentrations but at the highest concentrations i observed a significant decrease in the fluorescence intensity. This pattern is erratic and doesn't make any sense. Moreover, without adding inhibitor and using the same protein batch of holoprotein I see a consistent decrease in intensity on adding the quencher. Earlier I have observed the metal ions added tend to form some precipitates with the inhibitor. So take care of this issue I add minimal concentration of metal ion so that excess metal is less followed by filtration of the prepared holoprotein after adding inhibitor.
Hello Ankita,
as far as I understand, you have been using fixed concentration of protein and fixed concentration of inhibitor, but varying concentration of the quencher. Maybe you should try using different concentrations of the inhibitor as well? You could choose a couple of concentrations of the quencher (e.g., one on the slope and one on the plateau of FI signal) and then add increasing concentration of inhibitor to the mixture.
Hi Darja.
Thank you for the suggestion. you are right I am using a fixed concentration of encher. Conc of Inhibitor is approximately little higher than the IC50 value. If i vary the inhibitor how will i differentiate the quenching is due to the quencher or due to the inhibitor itself. Moreover, my reconsituted protein is in limited quantity due to technical constraints. another thing is that I have done lifetime meausrements at the above mentioned conc. of inhibitor. can I use the lifetime data and use it to calculate Kq from Ksv value obtained from this method.
I would refer to do the same at different temperature.. i.e 25 degree, 30 degree and 35 degree Celsius. such erratic pattern may come, if tryptophan is in close to the solvent.
Just do the experiment and lets chat later.
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