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Immunodetection

Western Blot

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Learn about the purpose of Western Blotting and the key steps involved. 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.

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 
  • 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

Obtaining and Recording Data

  • 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

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 Samuel Egieyeh
University of the Western Cape

"The Learning Science platform, with its interactive animations, provides the students with a basic understanding of the central dogma of molecular biology and provides the link between the basic biotechnology experiments and the complex production process, formulation, and quality control of biologics."

Details

Degrees

Biochemistry
Biomedical science
Biological science
Pharmacological science
Forensic science

Modules

Biological chemistry
Pharmacology

Topic

Immunodetection

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