What is western blotting?

Western blotting is a popular technique used for protein detection and quantitation. It enables the separation and identification of a specific protein of interest from a complex mixture of proteins, for example a cell lysate. With applications in diagnostics, biotechnology, molecular biology, proteomics, and much more, western blots are widely relied upon to evaluate levels of protein expression in cells, as well changes in size and other properties.

 

Western blot methods

There are several western blot methods to consider depending upon the secondary antibody used, detection of the target protein may be colorimetric, chemiluminescent, or fluorescent. These methods are described below and require different equipment for detection. For example chemiluminescence can be detected using x-ray film or digital imaging equipment while a fluorescent secondary antibody requires a fluorescence imager. Each type of detection has advantages and disadvantages that need to be considered when selecting a method.

Another method that we have introduced the ScanLater™ Western Blot Detection System, enables first-of-its-kind western blot detection in a multi-mode microplate reader platform. Utilizing this substrate-free assay, researchers can achieve equivalent sensitivity to traditional chemiluminescence assays while consolidating western blot, ELISA, and other applications onto a single reader. Our featured application notes below provide further details.

ScanLater Western Blot Detection Cartridge

ScanLater™ Western Blot Detection System enables western blot detection on microplate reader

 

Western blotting assay workflow

Here are the steps used for a typical western blot assay:

Western blot assay workflow

 

1. Perform electrophoresis – Proteins are first separated by size using gel electrophoresis.


2.  Transfer – They are then transferred to a blotting membrane, usually made of nitrocellulose or PVDF, which is probed with a primary antibody specific to the protein of interest.


3. Block with blocking buffer – This prevents binding of the primary antibody (next step) to the membrane itself.


4. Incubate with primary antibody – Primary antibody binds to the target protein.

5. Wash with wash buffer Unbound primary antibody is washed away.


6. Incubate with secondary antibody – A secondary antibody that recognizes and binds the primary antibody is added. This secondary antibody is conjugated to an enzyme or other material that allows detection of the protein of interest, which appears as a band on the blot.


7. Wash with wash buffer – Unbound secondary antibody is washed away.


8. Detect using method/chemistry of choice – Target protein can be detected by colorimetric, fluorescent, or luminescent methods, depending upon the secondary antibody used.

 

 

  • Colorimetric Western Blot

    Colorimetric Western Blot

    A colorimetric western blot method uses an enzyme-conjugated secondary antibody and a chromogenic substrate for detection.

    Advantages:

    • Require no special equipment

    Disadvantages:

    • Sensitivity is limited (pg range)
    • Blots cannot be stripped and reprobed to investigate additional targets

    Chemiluminescent Western Blot

    Chemiluminescent Western Blot

    A chemiluminescent western blot uses an enzyme-conjugated secondary antibody and a luminescent substrate. Results are detected using x-ray film and darkroom equipment, or a digital imaging system.

    Advantages:

    • Sensitivity (fg levels)
    • Blots can be stripped and re-probed

    Disadvantages:

    • Results can be non-linear
    • Signal has a short lifetime
    • Equipment required for detection can be expensive and space-consuming

  • Fluorescent Western Blot

    Fluorescent Western Blot

    A fluorescent western blot uses a secondary antibody conjugated to a fluorophore, so no substrate is required. A fluorescence imager is required to detect results.

    Advantages:

    • Greater dynamic range and better linearity than colorimetric or luminescent methods
    • Can multiplex using different fluorophores

    Disadvantages:

    • Can be less sensitive than chemiluminescence
    • Requires a dedicated fluorescence imaging system

    ScanLater Western Blot Detection System

    ScanLater Western Blot Detection System

    With the ScanLater Western Blot Detection System, secondary antibody is conjugated to a time-resolved fluorophore, combining advantages of both fluorescent and chemiluminescent methods.

    • No substrate required for detection
    • Signal stability of months or more
    • Sensitivity at sub-picogram levels
    • Wide dynamic range

    Download datasheet  

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  • Detection and quantitation of protein with ScanLater Western Blot Detection System

    Protein Detection ScanLater Western Blot Detection

    Protein detection is important for pharmaceutical and clinical research today, and western blots are among the most common methods employed for this purpose. Various techniques are used to detect proteins on western blot membranes including fluorescence and chemiluminescence. However, each technique has its limitations, and there is a continuing need to improve quantitation, accuracy, and dynamic range.

    This application note demonstrates extended dynamic range due to reduced background as well as stability of detection over time and number of reads. In addition, a comparison of Scanlater Western Blot System to a chemiluminescence method demonstrates improved sensitivity.

    Read application note  

    Validate CRISPR-Edited Cells using Imaging and Western Blot Detection

    Validate CRISPR-Cells using Imaging and Western Blot Detection

    Genome editing has been widely used to study gene expression and protein function, but many of these methods are labor intensive and inaccurate1 . The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has become a very popular tool for editing genes due to its high accuracy and ease of use2.

    Here, we demonstrate how the SpectraMax i3x Multi-Mode Microplate Reader and ScanLater Western Blot Detection System can be used to validate CRISPR/Cas9 genome editing.

    Read application note  

  • Wells to Westerns: Investigating the cellular heat shock response

    Wells to Westerns: Cellular Heat Shock Response

    Investigating a cellular response can involve multiple approaches to gathering information—imaging, cell-based viability and proliferation assays, western blots to look at changes in protein expression, and more. Often multiple instrument platforms are needed to glean the necessary results, and several software packages may need to be learned in the process.

    In this application note we show how data for several related cellular parameters was collected using a single instrument, the SpectraMax® i3 Multi-Mode Detection Platform.

    Read application note  

    Estimation of Protein Molecular Weights with ScanLater Western Blot Protein Ladder

    Estimation of Protein Molecular Weights

    The ScanLater™ Western Blot Protein Ladder is an essential component of the ScanLater™ Western Blot Detection System. The primary applications of this protein ladder include the estimation of protein molecular weight, the visualization of gel electrophoresis, and the evaluation of the transfer process from gel to blot.

    Learn how users can follow the electrophoresis and blotting workflow that is optimized for their application.

    Read application note  

  • Protein Detection Based on Europium Labeled Proteins

    Protein Detection on Europium Labeled Proteins

    Western Blot Protein Detection and Quantitation Based on Europium Labeled Proteins using a Plate Reader

    Here we report a novel system for western blot analysis that is incorporated into a SpectraMax® multi-mode microplate reader. Membranes are probed with europium labeled secondary antibodies or streptavidin that label the protein of interest. Europium (Eu) has a long fluorescence lifetime, on the order of 1 msec, and detection is done with time resolved fluorescence (TRF) detection mode, which significantly reduces background from auto-fluorescence or other sources of short lifetime emissions.

Resources for Western Blot

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ScanLater Western Blot Detection Cartridge

ScanLater™ Western Blot Detection System enables western blot detection on microplate reader

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