Lecture notes
Measuring Gene Expression and Western Blotting
- Institution
- The Manchester Metropolitan University (MMU)
Lecture notes on Measuring Gene Expression and Western Blotting
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https://www.youtube.com/watch?v=GJJGNOdhP8whttps://www.youtube.com/watch?v=IoVzpL_heFo
Protein level - Western blotting
Western Blotting (Immunoblotting): Principles
A laboratory method using antibodies to detect
specific protein molecules in a mixture of proteins.
Combines:
- Separation of proteins in a semi-solid
Polyacrylamid gel (PAG) using electrophoresis
- Electroblotting of the separated proteins onto a
solid membrane (nitrocellulose or
polyvinylidene fluoride (PVDF)
- Detection of target proteins by specific
antibodies
Gel Electrophoresis Formats
Sample preparation for SDS-PAGE
- Reducing agents (e.g. 2- mercaptoethanol): reduce
disulphide bridges and dissolve tertiary and quaternary
structures
- SDS: attaches to protein molecules, charges them
negatively and linearises the protein chains
SDS-PAGE
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