NUSCTX 170 Lecture Notes - Lecture 23: Polyvinylidene Fluoride, Nitrocellulose, Immunocytochemistry
3 Dec 2020
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Lab 23: western blot, standard biochemistry technique for quantification of protein expression in biological samples. Is there more protein in one set of samples compared to the other: similar to immunocytochemistry, uses antibodies to detect proteins of interest. In contrast to immunocytochemistry, biological samples are not fixed. Technical considerations: sds-page protein gels: % (lower % = looser, separation of higher molecular weights, transfer method: wet, semi-dry, or dry, membrane: nitrocellulose of pvdf, detection: fluorescent, chemiluminescent. Sds and beta-mercaptoethanol: beta-mercaptoethanol is a strong reducing agent reduces s-s bonds in proteins, sds is a strong detergent denatures proteins and solubilize hydrophobic pockets, proteins loose quaternary, tertiary and secondary structures. Sometimes higher background staining is seen with pvdf membranes: prepare transfer buffer equilibrate gels and membranes assemble the gel and membrane sandwich set up the transfer cell start the transfer. Gel transfer methods: wet vs semi-dry: following gel electrophoresis, the separated protein mixtures are transferred to a solid support for further analysis.
