Western blotting - a detailed guide

General procedure and tips for sample preparation, electrophoresis, transfer, and blotting.

Abcam app western blot protocol

Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis. The blot is a membrane, almost always of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed next to the membrane and application of an electrical current induces the proteins in the gel to move to the membrane where they adhere. The membrane is then a replica of the gel's protein pattern, and is subsequently stained with an antibody.

The following guide discusses the entire process of producing a western blot: sample preparation, gel electrophoresis, transfer from gel to membrane, and immunostain of the blot. The guide is intended to be an educational resource to introduce the method rather than a benchtop protocol, but a more concise document suitable for consulting during an experiment can be composed by selecting and editing relevant material. Consult the manufacturers of your electophoresis and transfer equipment for more detailed instructions on these steps.

  1. Sample preparation

    1. Lysis buffers
    2. Protease and phosphatase inhibitors
    3. Preparation of lysate from cell culture
    4. Preparation of lysate from tissues
    5. Determination of protein concentration
    6. Preparation of samples for loading into gels

  2. Electrophoresis

    1. Preparation of PAGE gels
    2. Positive controls
    3. Molecular weight markers
    4. Loading samples and running the gel
    5. Use of loading controls

  3. Transfer of proteins and staining (western blotting)

    1. Visualization of proteins in gels
    2. Transfer
    3. Visualization of proteins in membranes: Ponceau Red
    4. Blocking the membrane
    5. Incubation with the primary antibody
    6. Incubation with the secondary antibody
    7. Development methods

References

Harlow, Ed, and David Lane. Using Antibodies. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1999.

B.D. Hames and D. Rickwood. Gel Electrophoresis of Proteins: A Practical Approach 3rd Edition, The Practical Approach Series, Oxford University Press, 1998.

Bolt and Mahoney, High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate– polyacrylamide, gel electrophoresis. Analytical Biochemistry 247, 185–192 (1997).


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