BIOC 300D2 Lecture Notes - Lecture 10: Tissue Culture, Oncolytic Virus, Saponin
8 Jul 2016
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Technique that uses antibodies coupled (conjugated) with fluorescent dyes, such that you can observe proteins in a tissue, cells. Energy is absorbed by an atom which becomes excited. The electron jumps to a higher energy level. The electron drops back to the ground state, emitting a photon that you detect. The emitted light is always of lower energy (emission is always of longer wavelength) Conjugated to antibodies, always absorb higher energy and emit at lower energy. E. g. fluorescein (green), dapi (binds to dna, good for location of dna in sample and. In order to co-localize two proteins, we use two fluorophores that are conjugated to two antibodies. Fluorophores need to be different, need to separate the signal. Antibodies (immunoglobulins) are proteins produced by the b-cells and plasma cells. Most antibodies used in immunocytochemistry are of the igg class of immunoglobulins. Y-shaped molecules with two light and two heavy chains linked by disulfide bond, and two antigen-binding sites (variable portion).
