Dear colleagues and experts,
We were trying to conjugate an antigen with 48kDa with 40nm AuNPs.
We tried using 20ug of antigen (diluted in 20mM PB, 150mM NaCl,1‰P300 buffer) + 250ul of AuNPs OD20 + buffer of varying pH (6-10)
After incubating in a shaker for 10 minutes, we performed a salt test with 10%NaCl. All conjugates turned purple and precipitated.
How to troubleshoot this problem? Thanks in advance
You must not have enough protein molecules on the surface of the nanoparticles to render them stable, increase the amount of protein or decrease the volume of nanoparticles.
Thank you for the answer. We have tried using up to 100ug of antigen, salt test still crashed at all pH
Is it possible that the antibody/antigen used was not able to be conjugated by passive conjugation? If so, how to know if the antibody/antigen is able to be conjugated or not.
Proteins are polymers and as such they can provide steric stability, despite this, the hydrophobic patches of the protein molecules (once they are adsorbed) must be hindered enough to render the system stable. Antibodies are regularly adsorbed (passively) on the surface of gold nanoparticles, other proteins too. Tween 20 helps to reduce hydrophobic interactions, add it after your 10 min of adsorption.
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