Antibody is specific and monoclonal to the surface receptor of the mammalian cell.Antibody conjugated with gold nano particle which is citrate stabilized.After treatment of nano particle-Ab conjugate with cell i want to quantify the unbound Gold NP-Ab conjugate also.Can anyone suggest me how to collect the fraction of unbound Np-Ab conjugate.
your valuable suggestions will be highly obliged.thanks in advance
Dear Kumar, as I understand your question, you need to quantify the unbound conjugate in the supernatant. Supposedly you have added the labeled antibody to your cells after removing the medium, so you may use a photometer to assess the gold nanoparticle concentration against a standard curve generated from labeled antibody.
People use different inhibitor to stop the endocytosis process and later they collect the cell supernatant to measure the unbound particles.
thank you @ Wolfgang Schechinger.I am not fluorescently labelling the antibody.I have prpeared nano-conjugates(GOLD Np's with Antibody).Can you forward me further details or citation using photometer to assess the gold nanoparticle concentration against a standard curve generated from labeled antibody.
Thank you @Michael G. Weller .i have adherent cells .assay will be done in both the cases.Adherent as well as cell suspension.
If you are carrying out the binding experiment in both type of cells than in case of adherent cells you may pour out the supernatant from the wells after incubation and unbound may be read. Whereas in case of cell suspension you may centrifuge out the cells bound with Gold NP-Ab after incubation. Pour out the unbound and read it.
I'd just run a UV/Vis spectrum to identify an optimal wavelength for measuring the gold particles. Should be somewhere in green spectral range, but the exact value surely depends on the gold particle size and -distribution. Then generate a standard curve relative to the labeling solution and there you go.
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