I have experimented with imaging proteins (IgG, IgM) on mica and glass, both with treatments of APTES to generate AP-Mica and a glutaraldehyde treatment to expose reactive aldehyde groups on the surface in an effort to covalently link the protein to the surface. In both fluid and air imaging, I get sub-optimal adhesion of my proteins. They are not immobilized well enough to get high resolution sub-structure of the antibodies (they move too much). Are there any better substrates/treatments to use when working with proteins in this size range (15-30 nm) for either air tapping mode or fluid tapping mode?
I haven't worked much with these specific proteins, but I had pretty good success in the past with using HOPG as a substrate for some proteins. I found a paper in Journal of Colloid and Interface Science, 166(1), 1994, Pages 102–108, stating that the authors managed to successfully do AFM imaging of IgG adsorbed on HOPG in a native conformation. Perhaps something for you to try?
Ricardo Garcia's group at IMM Madrid did quite a bit of AFM imaging of antibodies in air, e.g., BiophysJ 78 (2000) 1599–1605, Nanotechnology 19 (2008) 384011. Their papers deal more with the fine control and tracking of the cantilever dynamics than actual biology, but perhaps something could be of help there.
I don't think they shud b moving . I have used these surfaces for real small proteins and never had any problem . Can u tell me how u r preparing the surfaces and samples
Recently, I tried again for IgG on AP-Mica, and succeeded only after reducing my free amplitude greatly to reduce tip forces on the sample. I will re-attempt on regular mica with the lower amplitude. I believe that is what my problem was, thank you all for the help.
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