I am working on a project to synthesise molecularly imprinted polymers to a protein that is bound to the surface of silica nanoparticles using glutaraldehyde. I want to make sure that the template protein is attached to the surface of the particles before polymerisation, The traditional BCA test appears to react with glutaraldehyde giving false positive results. Acid Violet test gives marginally better results. Do you know of any other test that doesn't interact with glutaraldehyde?
Thank you.
Siva
Hi Muttuswamy, if you are not using aminosilanes to coat your silica nanoparticle before the glutaraldehyde treatment then nothing will happen.
Silica has exposed -OH groups. These do not react with aldehydes -CHO.
If you are seeing proteins on the particles, then it is probably just aggregation of crosslinked proteins on the particles.
Knowledge of silane chemistry is essential when you are working with silica.
Thank you for your contribution. We are following Pilersky et al protocol published in Nature Protocols for MIP synthesis. Glass beads were treated with APTMS prior to protein immobilisation (glass beads - NaOH- APTMS - glutaldehyde - template binding-polymerisation)
My questions is this- what's the best test to demonstrate protein binding to glutaraldehyde treated glass nanoparticles?
Kind regards
Siva
OK. Glutaraldehyde is not the best way to bind proteins to APTMS treated silica. Glut. binding to -NH2 is reversible and is dependent on the pH and species of buffer used.
If your buffer has primary or secondary amines, or a pH above 8.0, the coupling will not work well.
My recommendation is to use EDC x-linking in MES buffer at pH 5.5. EDC activates the -COOH groups of the proteins and they will link to the -NH2 on the silica. A caution though, incubate the silica with EDC and add the protein in gradually. .
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