Hi all,
I'm trying to doing a fluorescence screening to determine protein/ligand binding using an extrinsic fluorophore --ANS. Referring to an artical published, controlled lighting was used to stabilise the fluorescence of ANS ~1h, then different compounds were added. I don't really understand what the "controlled lighting" can do here. Can someone explain it? Many thanks!
Hi, I am also working with ANS as fluorescence probe for protein-ligand binding analysis . I think that "controlled lighting" could be made because perhaps the ligand is light sensitive, so that the ligand must be protected of light. However, I agree with Vivek and I would like to see the article.
Many fluorescent compounds can be damaged by excessive exposure to light. Normally, one tries to minimize this exposure to avoid photobleaching,
Hi all, thank you for your explanations. I have tried to use ANS with my protein but it seems the fluorescence signal only increased by 20 (from 200-220) after the addition of protein, which probably means ANS didn't interact with the protein.
Could you give me some advice on the following question please?
1. Could I prepare ANS stock solution in DMSO?
2. Did you test the fluorescence right after the addition of protein?
Thank you in advance!
ps. the link of the paper is attached
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3483082/
ANS will only increase in fluorescence mostly if it is bound to a hydrophobic environment. However if it is bound to some solvent exposed site through some electrostatic interaction then it may not increase in fluorescence significantly. Fluorescence increase is a good test for the presence of hydrophobic surfaces, but maybe not so much for binding.
A 20 fold increase may mean that your binding site may not be too solvent restricted. It could also mean that you did not saturate your protein as mentioned above. You may gain more information by looking at the wavelength of the emission maxima as well.
DMSO stocks are fine as long as your protein can tolerate it. I like to use buffer stocks to avoid complications, often I can get 10mM in solution for 18ANS.
ANS is fairly stable, as long as protein is stable you should have the same results within a couple of days. I have observed strong changes in absorption of buffered stocks after 1 year in the fridge in the dark.
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