Immunodetection

Western Blot

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Explore how proteins can be transferred to a porous membrane and detected using antibodies. Practise setting up the apparatus for a wet transfer and interpreting the bands produced after blotting.

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Learning outcomes

Familiarise yourself with the purpose of a western blot.
Explore the theory of the key steps.
Learn how to set up a western blot transfer (specifically a wet transfer) and probe for a particular protein.

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Skills

Concepts, Principles and Theories

How antibodies can be used to detect the presence of target protein within a sample
What is happening on a molecular level during each step of the immunoblotting process

Using Instruments and Equipment

How to set up the blotting sandwich
How to set up the transfer apparatus
How to label the membrane with primary and secondary antibodies
How to detect the presence of bound antibodies

Obtaining and Recording Data

Why the order of the blotting sandwich components is important
Why the orientation of the blotting sandwich in relation to the electrodes is important
Why buffer, a stir bar and an ice pack should be included in the transfer apparatus
Why including blocking and wash steps improves the sensitivity of the technique
Why a loading control is required

Designing and Planning Experiments

How to choose the right secondary antibody to pair with the primary antibody

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Interpreting and Analysing Data

How the results of a western blot can be used to confirm the presence of a protein in a mixture of proteins
How a western blot can be used to compare the level of protein between samples

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Dr Anna Maria Piccinini

University of Nottingham

"Always accessible everywhere, these resources have added flexibility to PGR training, allowing students to safely prepare for the lab and regain self-confidence"

Details

Subject areas

Biological science

Biomedical science

Forensic science

Pharmaceutical science

Discipline

Molecular biology

Topic

Immunodetection

Purchase option

Bioscience LabSim Library

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