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.

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. In direct 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 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! Click here to see our full range of Lightning-Link® Antibody Labelling kits which include our enzyme labelling kits for Horseradish Peroxidase and Alkaline Phosphatase and our fluorescent dyes.

RunBlue FAST Blotting Buffer

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

  • Reduce 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:

Western blot - Enzyme substrate direct and indirect method

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:

Western blot - Fluorescence direct method

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. For more information download our Western blot Guide.

 

 

 

 

Chemiluminescent Western Blotting:

Western blot - Chemiluminescence direct method

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 our Western blot Guide.

 

 

 

 

For more information download our Guide to Western blotting.

 

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