Hi ladies and gents. I'm trying to detect a fluorphore-conjugated antibody linked to a solid surface using a synergy plate reader. My question is: would I be better off running the analysis in wells with or without a buffer solution?
I am concerned on the one hand that the buffer would quench the emission from the object in the bottom of the well. On the other hand, I don't know if the antibody/fluorophore being dry will denature it to the point that it will no longer fluoresce.
Any ideas?
If your conjugate is perfectly immobilized ..you may not need any buffer.
The conjugate should be covalently bound to the surface... I'm just concerned that some denaturation may take place if the protein dries out.
For a simple detection, measurement without buffer should be sufficient.
If you aim at quantification or very sensitive detection, you have to take into account the nature of the fluorophore. For instance, fluorescence emission of fluoresceine changes drastically with respect to the actual pH. Therefore, depending on your specific fluorophore, I recommend to use a buffer.
Hello! If you think about antibody arrays being sold by many companies with varying immobilisation techniques and shipped dry, you have an example that it will be fine. However, every antibody is different and conjugation to either the surface or dye alters its behaviour in the sense of susceptibility to denaturation or decrease in affinity to target.
My experiments with Pherastar showed 5-20% increase in intensity if buffer was removed. Your Synergy equipment might have the possibility to measure intensity from the bottom, therefore buffer would not diminish the signal relative to dry (considering clear bottomed well and loss in intensity to plastics/glass environment). Dry fluorophores might be destroyed faster relative to the ones in solution.
I am very interested, if you have coupled the antibody to the surface by yourself and if yes, by what means?!
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