I need to study interaction with a drug with BSA,but here my drug is also florescence active.I am used to study quenching of BSA with Drug,where i kept BSA as a fluorescence active,but i have faced some problems,when i seen DE-quenching due to interaction of drug with BSA .It means, the fluorescence absorption is increasing with the increase of drug concentration.,i have used 20uM (BSA fixed) and drug concentration increase from 20uM to 320uM concentration.
It's difficult to say without the knowledge of the absorption and fluorescence band positions of the drug you are using.
I can not understand as you mentioned about de-quenching due to BSA-drug interaction and also that fluorescence intensity is increasing upon increasing of drug concentration? By the way, in both cases changes could be as a result of interaction. As I see your maximum position is fixed at around 338 nm. To study BSA interactions using fluorescnece capacity of the drug you really need to know its absorption and fluorescence max positions....
Dear, Lidiya Petrova.
As i used BSA as fluorescence active molecules so i have focused on BSA only,i used two excitation wavelength for BSA (excitation wavelengths are,at 280nm & 293nm) From the spectral analysis i have found that Fluorescence emission is increasing with increasing of Drug concentration.But comparing my previous run experience i have figured that,Fluorescence emission were decreased with the increase of drug concentration.So,now i am searching for the method to calculate my data in such situation when BSA absorption or fluorescence emission is increase with the increase of concentration of Drug.
Dear Rupashree Balia Singh,
Do i need to know the wavelength position and absorption of my drug molecules ?,If i found the position, then how do i measure my drug interaction with BSA ? do i need to use fixed Drug concentration Vs variable BSA concentration ? .what will be the process ?,if you share it then it will be convenient for me.
Yes, I think that information is very crucial for you to be able to decipher your findings.
Say, e.g. your drug and BSA have overlapping absorption and emission bands. In that you might be exciting both and the increase in emission could be some strange misleading observation and not the result of any interaction. You may measure the excitation spectrum corresponding to emission to confirm what you are observation.
FRET from drug to BSA is another possibility but, I think that is unlikely. But, you can still try looking at the drug fluorescence by varying BSA conc. You have to try various permutation and combination to come to a conclusion.
I think as the first step you must known the absorption and emission band positions of the drug. This will help you think further. Go one step at a time.
Good luck !
maybe your can go on the reaction of drug with BSA, control the concentration both of them, then along the reaction time. another method is Synchronous fluorescence method. good luck!
After you finish what was mentioned by others, you can also try fluorescence correlation spectroscopy (FCS)
You can follow Monica Tory's Process. It will be helpful for you.
Good luck!
Such fluorophore-BSA interactions are ofted studied using fluorescence anisotropy (time-resolved if uou have access to such equippment) or even at the cell level using confocal microscope (FCS, FLIm). It depends what kind of information you need from your measurement.
Good luck
In addition to the above, fluorescence polarisation could be used to measure any binding that may occur. The value of the anisotropy should increase due to increased mass of BSA binding to your molecule. Standard fluorometers can be upgraded relatively cheaply if they don't already have polarisation filters installed.
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Sometimes difference spectroscopy provides a sensitive and useful method to study interactions between two molecules. Difference spectroscopy does not require the molecules to be fluorescent. What you need are a reasonably good dual beam spectrophotometer and a special set of cuvettes that are divided into two compartments. In one cuvette, you place substance 1 into compartment A and substance 2 into compartment B. In the second cuvette, you fill both compartment A and compartment B with a mixture of substance 1 and substance 2. You then place both cuvettes into a dual beam spectrophotometer in such a way that the light beams transsect both compartment A and compartment B. You then record a spectrum. If substance 1 and substance 2 do not interact with each other, you will get a flat line because sample beam and reference beam are recording the same spectrum. If substance 1 and substance 2 do interact, sample beam and reference beam will record two different spectra, and the difference will be displayed by the instrument. The technique is very sensitive. Even minor spectral differences generate large signals.
That's a primitive explanation of the technique. If you search "difference spectroscopy" in Medline or Google, you will get plenty of hits, including examples of difference spectra.
Thank everyone for solutions.I hope i can find some thing new to start with.Thank again.
Please Check following link, may help you
http://pubs.rsc.org/en/content/articlelanding/2014/pp/c4pp00099d#!divAbstract
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