Introduction to Western Blotting


Western Blot Learning Center

Instruction for all steps in the western blotting workflow.

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Western blotting is a powerful technique that allows you to positively detect your proteins, estimate quantities, and determine their molecular weights. All from a starting mixture of proteins extracted from cells or tissues.

The entire western blotting workflow consists of several individual steps, each of which is critical to producing high-quality data.

This Learning Center provides in-depth information on the theory and practice of each step of the western blotting process, starting from sample preparation to analysis of the final blot

  • Sample Preparation

    Sample Preparation

    Cells containing your protein of interest must be lysed completely to ensure a high yield while removing non-protein components of cells. Good sample preparation techniques ensure proteins remain undamaged for downstream analysis.

  • Electrophoresis


    Electrophoresis separates the proteins in the sample and provides molecular weight data for detected proteins during subsequent detection.

  • Transfer


    Separated proteins are transferred from the gel to a membrane where they are immobilized. Efficient protein transfer is required for maximum western blot sensitivity.

  • Detection


    A good, clean western relies on the specificity and sensitivity of your antibodies. In addition to detection of specific proteins, the total protein in a sample can also be visualized with total protein stains or newer technologies that eliminate the need for staining and destaining.

  • Image Acquisition

    Image Acquisition

    Accurate imaging of your western blot is crucial for capturing blot data for downstream analysis. Understanding basic imaging concepts such as sensitivity, resolution, and sources of background noise can help you maximize image data quality.

  • Image Analysis and Quantification

    Image Analysis and Quantification

    An image of a western blot is rich with information. Some experiments require only a qualitative answer, but properly designed and executed western blot experiments can also provide quantitative data on relative protein expression between samples.


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