MIC 410 Lecture Notes - Lecture 21: Horseradish Peroxidase, Polyvinylidene Fluoride, Primary And Secondary Antibodies
10 Dec 2019
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Indirect: antigen, primary anitbody, secondary antibody, enzyme, substrate. Coat the wells with samples and controls. Coating buffer: helps stabilize the protein (antigen/antibody) so it sticks to the wells. Sandwich: capture antibody, antigen, primary anitbody, secondary antibody, enzyme, substrate. Goal for our elisa: detect tsh (temperature sensitive hemagglutinin) in our unknown samples. Run an sds-page ( to separate proteins) Stacking gel: on top; lower conc; makes resolution clear. Resolving gel: on bottom; higher concen; separates proteins. Made of nc or pvdf (for hydrophobic) Milk-ttbs solution prevents the antibodies from getting stuck on the membrane. Protein samples are mixed with sample buffer. Sds: anionic detergent that denatures proteins and gives the proteins a uniform negative charge. As a result, proteins are made linear thus separating solely by size. Glycerol: provides the sample density so that it does not float out of the wells when loading into the gel. -mercaptoethanol: a reducing agent that breaks down disulfide bonds.
