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Western Blot

The aim of Western blotting is to identify specific proteins within a complex mixture. The Western blot technique requires samples to be resolved on the basis of size through Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), following which they are transferred to, and immobilized on, a membrane prior to antibody-based detection.

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Western Blot Indirect and Direct Labeling

Before running a Western blot it is extremely important to research the target protein thoroughly. To know more about this, refer to our Western blot guide.

  1. In a traditional Western blot -indirect labeling-, protein samples are first resolved by SDS-PAGE and then electrophoretically transferred to the membrane.
  2. Subsequent to a blocking step, the membrane is probed with a primary antibody (poly- or monoclonal) that was raised against the antigen in question.
  3. Following a washing step, the membrane is typically incubated with a dye or enzyme-conjugated secondary antibody that is directed against the primary.
  4. The fluorescence of the dye or activity of the enzyme, such as Alkaline Phosphatase (AP), Glucose Oxidase (GO) or Horseradish Peroxidase (HRP) is necessary for signal generation.
  5. Finally, the membrane is washed again and incubated with an appropriate enzyme substrate (if necessary), producing a recordable signal.
  6. Indirect analysis, the need for the secondary antibody step is eliminated thereby simplifying the procedure, shortening the protocol and expediting the time to results.

Please watch our on-demand Western blot Webinar for more information on the use of directly labeled antibodies for Western blotting.

 

Lightning-Link® – the world’s easiest antibody labeling technology for direct detection

Our Lightning-Link® antibody and protein labeling kits allow researchers to directly label their antibody, eliminating the need for a secondary antibody. The kits require only 30 seconds of hands-on time, saving valuable time in the lab. Furthermore, there are no separation steps involved post conjugation, so you will retain 100% of your antibody! Discover our full range of Lightning-Link® Antibody Labelling kits which include our enzyme labeling kits for Horseradish Peroxidase and Alkaline Phosphatase and our fluorescent dyes.

 

RunBlue FAST Blotting Buffer

Our RunBlue FAST Blotting Buffer increases the transfer speed of Western Blots without excessive heat generation that affects proteins.

  • Reduces blotting time by up to 60%
  • Smaller protein transfer in less than 10 minutes and larger protein in only 15 minutes

Check our range of Western blotting products from Expedeon that can assist you further.

 

Immunoblot detection

There are several different choices of readout when Western blotting. Each with advantages and disadvantages – depending upon what your needs are and what equipment is available in your lab.

  • Colorimetric blotting.
  • Fluorometric blotting.
  • Chemiluminescent blotting.

 

Colorimetric Western Blotting

Schematic Representation of colorimetric Western blot detection

Figure 1. Schematic representation of colorimetric Western blot detection. The upper panel demonstrates indirect detection while the lower panel shows direct detection. Indirect versus direct detection is discussed in more detail within our Western blot guide; Lightning-Link® facilitates direct detection.

Colorimetric detection relies on the generation of a colored product that becomes deposited on the Western blot; this is formed following the conversion of a chromogenic blotting substrate by an appropriate enzyme. The limited sensitivity of chromogenic substrates can make it difficult to optimize them for detecting proteins of low abundance; although the chromogenic reaction can be allowed to develop for several hours (or even overnight) this allows background signal to develop simultaneously.

Colorimetric substrates are however perfect for the detection of abundant proteins since the reaction can be monitored visually and allowed to progress until the color development is adequate before being stopped. No specialized equipment is required for visualization of the colored precipitate, and the signal that is produced is highly stable. For more information download our Western blot Guide.

 

Fluorometric Western Blotting

Schematic representation of Western blot fluorometric detection

Figure 2. Schematic representation of fluorescent Western blot detection using Lightning-Link®

Fluorometric detection requires the use of an antibody which has been labeled with a fluorophore. A light source is used to excite the fluorophore, which then produces a transient light emission as it returns to its ground state. The light is emitted at a higher wavelength than that which was used for excitation and is detected with a specialized reader.

 

Chemiluminescent Western blotting

Schematic representation of antibody chemiluminescent Western blot detection using Lightning-Link®

Figure 3. Schematic representation of chemiluminescent Western blot detection using Lightning-Link®

Chemiluminescence occurs when a substrate is catalyzed by an enzyme and produces light as a by-product of the reaction. The limiting reagent in the reaction is the substrate; as this is exhausted the light production decreases and eventually stops, however, a well-optimized procedure should produce a stable light output for several hours allowing consistent and sensitive protein detection.

For more information:

Download guide

 

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A-Z Guide to Fluorochromes

August 23rd, 2017

        Download a Free Copy Lightning-Link® fluorescent antibody and protein labeling kits allow you to covalently label any antibody in under 20 minutes with only 30 seconds hands-on time. We currently have over 35 label in our range of fluorescent dyes. This guide provides a general overview of each fluorochrome in our…

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Absorption-Emission Table

August 23rd, 2017

Lightning-Link® is an innovative technology that enables direct labeling of antibodies, proteins, peptides or other biomolecules with only 30 seconds hands-on time.

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Simplifying Antibody Conjugation Process

August 23rd, 2017

Antibodies are widely employed in the quantification of antigens in complex biological samples. Using techniques such as Western blotting, ELISA, and immunohistochemistry researchers are able to measure a single antigen, or perhaps a limited number of antigens, in each sample. In the post-genomics era, advances in multiplex immunoassay technologies now allow scores or even hundreds…

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Antibody Purification Guide

August 23rd, 2017

        Download your copy Expedeon specializes in easy to use bioconjugation kits which enable the direct labeling of antibodies or proteins with enzymes, fluorescent labels, biotin, streptavidin, gold nanoparticles, latex beads or oligonucleotides. Unfortunately, many antibodies are provided in buffers which contain additives that are incompatible with labeling technologies, making puri cation…

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Guide to labeling your primary antibody

August 23rd, 2017

        Download a free copy! Antibodies are used to detect and quantify antigens in techniques such as flow cytometry, ELISA, western blotting, immunohistochemistry and lateral flow. The antibody that binds to the antigen is called a ‘primary antibody’ and it confers specificity to the assay. Most types of immunoassays also incorporate a…

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Guide to Western blotting

August 23rd, 2017

        Download a free copy The Western blot technique requires samples to be resolved on the basis of size through Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), following which they are transferred to, and immobilized on, a membrane prior to antibody-based detection. Find out how we can help you with your Western…

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Antibody labeling protocol video

May 7th, 2018

Applications, Videos

Stop using secondary antibodies in immunoassays! Save time and materials by labeling your primary antibodies in under 20 min. For use in ELISA, western blotting, IHC, flow cytometry and many other applications. Scientists love our Lightning-Link® kits because of the remarkable simplicity of the technology. Now they can open a…

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Protein labeling and conjugation

August 25th, 2017

Videos

Lightning-Link® LAMP1 labeled antibodies (MCF-7 human breast cancer cells). Nuclei stained with DAPI (blue) and LAMP1 antibody conjugated with Atto633 (red).

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Methods of antibody conjugation – which one is best for you?

January 20th, 2014

Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with their company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   Dr Nick Gee, the CEO and CSO of Expedeon, explains the benefits of using directly conjugated antibodies and the various approaches and techniques…

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How to Overcome all your Problems with Secondary Antibodies

December 6th, 2012

Applications, Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with our sister company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   This webinar discusses how to overcome problems associated with using secondary antibodies in your immunoassay, including: Secondary antibodies require a series of…

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Western Blotting – The principles and a comparison of indirect vs. direct immunodetection

October 3rd, 2010

Applications, Webinars

**This webinar was produced by Innova Biosciences which is now fully integrated with their company, Expedeon Ltd., and have taken the Expedeon Ltd name and logo.   Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture. Although Western blotting…

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