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.
||Normalization or comparative analysis is enabled by the infrared western blot without the stripping and reprobing of blots.
||Indirect (Enzymatic reaction)
||The IRDye® secondaries’ signal is directly proportional to the amount of target protein. In contrast, the chemiluminescence enzyme or substrate kinetics could affect performance.
||Months to years
||IRDye® fluorescent signal is very stable, therefore blots can be stored and re-imaged later.
||♦ ♦ ♦ ♦ ♦
||♦ ♦ ♦ ♦ ♦
||Infrared detection is more sensitive than other fluorescent methods and is equivalent to chemiluminescent methods.
||♦ ♦ ♦ ♦ ♦
||The IRDye® secondaries’ wide dynamic range saves time by reducing the need for multiple exposures.
||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.
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.
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