why do we use multiple concentrations of 0.05, 0.10, 0.15 and 0.20 mg/ml?
after i get the overall data of scanning, what is the next step to get the final results for hydrophobicity? I am using ANS.
I will be thankful if someone could explain this please.
Dear Ziaur Rahman Have a look at the following paper which perfectly explains why (you need these duplicate amounts for the subsequent calculations):
Article Spectrofluorimetric Assessment of the Surface Hydrophobicity...
Personally I would take the results of these type of assays with caution. At best it is indicative (especially when you compare it with some known examples described in the literature and preferably you measured yourself).
Do you know the exact composition of your protein? If so then I would consider bioinformatics to get idea's about hydrophobicity, type of protein (in terms of function), secondary structure etc.
Best regards.
To measure the surface hydrophobicity of a protein sample with a fluorescence spectrometer, you'll typically use a fluorescent probe like 8-anilino-1-naphthalenesulfonic acid (ANS). ANS is a dye that has a weak fluorescence in water but exhibits strong fluorescence when it binds to hydrophobic regions on the protein surface.
you can find step-by-step guide to perform the experiment and analyze the data?
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