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Lecture notes

Measuring Gene Expression and Western Blotting

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2021/2022 Written for academic year
Institution The Manchester Metropolitan University (MMU)
Module Techniques and Applications in Biomedical Science (6H5Z1036)
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Lecture notes on Measuring Gene Expression and Western Blotting

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https://www.youtube.com/watch?v=GJJGNOdhP8w
https://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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The Manchester Metropolitan University
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2021/2022
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Dr james pritchett
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