IRDye® conjugated secondary antibodies
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Learn about the advantages of using fluorescent western blot versus chemiluminescence and the value of IRDye® conjugated secondary antibodies for multiplex fluorescent western blot.
Western blot fluorescence vs chemiluminescence
Learn the advantages of IRDye® secondary antibodies that allow you to perform multiplex western blot and detect more than one protein in the same blot at the same time using IRDye® 800CW and IRDye® 680RD secondary antibodies. Get IRDye® secondary antibodies, then just follow our optimized fluorescent western blot protocol and detect your protein of interest.
Chemiluminescence | Fluorescence | Advantages | |
Multiplexing | No | Yes | Infrared western blot allows normalization or comparative analysis without stripping and reprobing of blots. |
Detection | Indirect (enzymatic reaction) | Direct (fluorescent) | When using IRDye® secondaries, signal is directly proportional to the amount of target protein, while in chemiluminescence enzyme/substrate kinetics may affect performance. |
Stability | Hours | Months to years | IRDye® fluorescent signal is highly stable, so you can store blots and re-image later. |
Sensitivity | ♦ ♦ ♦ ♦ ♦ | ♦ ♦ ♦ ♦ ♦ | Infrared detection is more sensitive than other fluorescent methods and equivalent to chemiluminescent methods. |
Time | ♦ ♦ | ♦ ♦ ♦ ♦ ♦ | The IRDye® secondaries wide dynamic range saves you time by reducing the need of multiple exposures. |
Secondary antibodies | HRP or AP conjugated |
Get IRDye® secondary antibodies
See below an example of how to do normalization against the housekeeping gene using IRDye® secondary antibodies
With IRDye® secondary antibodies in your fluorescent western blot, you can do normalization with an internal control in the same blot, without the inconveniences of stripping and reprobing again. Using IRDye® secondary antibodies in multiplex fluorescent western blot also allows sensitive and quantitative detection of your protein of interest if needed.
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 a 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 | |
Protein of interest | |||
Housekeeping gene (control) |