I have been having problems when trying to perform a bis-ANS/Cyp A assay.
Because bis-ANS is such a non-specific binder (binds to any hydrophobic patches on the protein) it should give a very nice binding curve in the presence of CypA as we are increasing its concentration.
Any suggestions as to why there is no change in fluorescence both in the presence and absence of protein?
(Have checked the excitation of bis-ANS and it everything is as it should be, so the problem is not the dye)
Many thanks
Since you did not specify the concentrations you use, my first guess would be the dye/protein ratio. I experienced that even minute quantities of ANS lead to a remarkable increase in fluorescence emission. So I would assume your assay lacks sensitivity because of excess dye.
Try the following: Take a 20 µM solution of your protein of interest (POI) and titrate that with a 2 mM solution of bis-ANS (better: ANS) in steps of 2 µl, to check on the responsiveness of the assay, which depends on your specific POI. This approach also gets yout the kD of your POI for the dye (assuming the simplified case of only one binding site).
Using ANS instead of bis-ANS most likely will lead to an increased sensititvity of the assay, since ANS only has (obviously) only half the chromophors bis-ANS has. Because of this, I have always prefered ANS over bis-ANS.
Greetings to Scotland and good luck!
thanks for your comment!
I've been using various concentrations of bis-ans: 0.1uM to 50uM, keeping cypA at a constant 1uM conc.
There is only the slightest change in fluoresence, about 1000cps. I've also used ANS and have gotten the same result.
So according to your comment, I may be using too little of the dye :s
Considering the concentrations, this would be highly probable. I always found it useful to perform a titration of the POI against ANS to get a feeling for what's happening.
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