We are trying to know if our protein of interest sticks to the material that we want to use. We tried to do an immuno assay with gold bound antibodies but we didn't see anything, even in the material that literature says that it sticks to (they measure the difference in the concentration of the protein in the medium, but we don't have a reliable way to measure it). ¿Does anyone have any idea?
Thank you
It would be helpful to know the kind of material. Are you talking about solid surfaces, a powder, nanoparticles? In general, difference measurements are not very useful, if you want to measure trace amounts, because the difference is negligible.
Maria, direct staining with gold bound antibodies is rather insensitive, but you could try an ELISA like procedure where peroxidase labelled antibodies come into play. For example, add HRP labeled antibodies against the gold labelled species, which might be the simplest approach.
There is some chance that the binding to the surface renders your protein inaccesible for the antibodies, so a negative result might be not conclusive, while, if it works, you even might be able to generate quantitative data (by including samples for a standard curve).
Otherwise, extraction with Laemmli Buffer like a standard sample for SDS-PAGE and then performing a western Blot for your target protein might help.
Michael G. Weller They are solid surfaces. For example what we were thinking to use as positive control is a piece of a polystyrene eppendorf. Thank you
Wolfgang Schechinger Great ideas, thank you¡ We will try with HRP labeled antibodies, but without the gold bound antibodies because we used them because our initial idea was to see it by SEM.
Western blot is a good idea too, but maybe we won't have enough protein.
Thank you so much¡¡
María Almudena Coto Vilcapoma . Please allow a short comment on your reply, "we were thinking to use as positive control is a piece of a polystyrene eppendorf".
Eppendorf tubes consist of polypropylene and today, many of them are produced with a "low-binding" surface. Therefore, they would be more suitable as a negative control.
In addition to the earlier suggestions, if you have access to ToF-SIMS capabilities, this would also allow you to understand the propensity of the surfaces to specific protein adsorption. True quantification is challenging, but not impossible. Altnernatively, the substrate may allow XPS surface elemental quantification, thus quantifying proteins on polyolefins via N-Amide signals.
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