The Advantages of the IRDye® Secondary Antibodies

The following article presents information about the infrared fluorescent western blot, including the multiplex western blot, that uses IRDye® conjugated secondary antibodies. The advantages of using IRDye® conjugated secondary antibodies over HRP secondary antibodies, which are used in chemiluminescence (ECL), are shown in the tables below.

Western Blot Fluorescence vs. Chemiluminescence

The IRDye® secondary antibodies have several important advantages. For instance, due to the multiplex western blot, they can detect more than one protein in the same blot at the same time using the IRDye® 800CW and IRDye® 680RD secondary antibodies.

The IRDye® secondary antibodies can help to detect a protein of interest by following the optimized western blot protocol.  

Chemiluminescence Fluorescence Advantages
Multiplexing No Yes Normalization or comparative analysis is enabled by the infrared western blot without the stripping and reprobing of blots.
Detection Indirect (Enzymatic reaction) Direct (Fluorescent) The IRDye® secondaries’ signal is directly proportional to the amount of target protein. In contrast, the chemiluminescence enzyme or substrate kinetics could affect performance.
Stability Hours Months to years IRDye® fluorescent signal is very stable, therefore blots can be stored and re-imaged later.
Sensitivity ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ ♦ Infrared detection is more sensitive than other fluorescent methods and is equivalent to chemiluminescent methods.
Time ♦ ♦ ♦ ♦ ♦ ♦ ♦ The IRDye® secondaries’ wide dynamic range saves time by reducing the need for multiple exposures.
Secondary antibodies HRP or AP conjugated secondaries IRDye® conjugated secondaries

Specifics of the IRDye® Secondary Antibodies

Below is an example of how to achieve normalization against the housekeeping gene by using IRDye® secondary antibodies.

The IRDye® secondary antibodies allow for normalization to be achieved in the fluorescent western blot with an internal control in the same blot. The inconvenience of stripping and reprobing is eliminated.

Sensitive and quantitative detection of the protein of interest is also achievable with the use of the IRDye® secondary antibodies in fluorescent western blot for multiplex western blot.

p53 analysis by infrared fluorescent western blot using anti-p53 antibody [DO-1] (ab1101) as primary antibody and goat anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) as the secondary antibody. Lane 1: Wild-type HAP1 cell lysate, lane 2: p53 knockout HAP1 cell lysate, lane 3: A431 cell lysate, lane 4: Saos-2 cell lysate.

Figure 1. p53 analysis by infrared fluorescent western blot using anti-p53 antibody [DO-1] (ab1101) as primary antibody and goat anti-Mouse IgG H&L (IRDye® 800CW) (ab216772) as the secondary antibody. Lane 1: Wild-type HAP1 cell lysate, lane 2: p53 knockout HAP1 cell lysate, lane 3: A431 cell lysate, lane 4: Saos-2 cell lysate.

Detect Primary antibody Secondary antibody
IRDye® 800CW

Protein of interest

Anti-p53 antibody [DO-1] (ab1101)

Goat anti-Mouse IGG H&L (IRDye® 800CW)-ab216772
IRDye® 680RD

Housekeeping gene (Control)

Anti-GAPDH antibody [EPR16891] (ab181602)

Goat Anti-Rabbit IGG H&L (IRDye® 680RD)- ab216777

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: May 3, 2019 at 4:34 AM

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