What do you mean "it settled down?" If you see black particles i guess you are using actual ANS and not its salt. Try using ANS amonium salt and disolve it in PBS buffer (ph 7.4) it should work.

yeah i do see black particles in that and settling down means its getting accumulated at the bottom of centrifuge tube and i did use actual ANS

Right, so do as I said: Try using ANS amonium salt and disolve it in PBS buffer (ph 7.4) it should work. Add it to protein solution 1:2 or 1:4, should work either way :)

ANS ammonium salt: https://www.sigmaaldrich.com/catalog/product/sigma/10417?lang=en®ion=SK