NUSCTX 170 Lecture Notes - Lecture 21: Immunolabeling, Primary And Secondary Antibodies, Immunochemistry
3 Dec 2020
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Lab 21: immunolabeling techniques, used to analyze cellular and tissue distribution of proteins, glycans, and small biological and non-biological molecules. Icc/if fluorescent dyes are invisible unless a light is shined on these dyes, can absorb and emit in a different wavelength. Need a fluorescent microscope (special that has a source of light very specific to emit different wavelengths and camera to capture), multiple colors. If the binding of the dye is so strong for the antibody, then may interfere with the antibody specificity. So the fluorescent can attach to the fab, then won"t be recognized. Indirect (like in lab) primary antibody is not fluorescently labeled. Instead, a secondary antibody is used which recognizes the fc domain of the primary antibody, and does not recognize the antigen. In indirect detection, you can amplify the signal: advantage of using fluorescently secondary antibody is signal amplification: several secondary antibodies can bind to the primary antibody and increase signal intensity.
