IRDye® conjugated secondary antibodies
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Learn about the advantages of infrared fluorescent western blot compared to chemiluminescence (ECL), including the ability to detect more than one protein in the same blot at the same time.
Advantages of infrared fluorescence over chemiluminescence
Chemiluminescence | Infrared 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) | 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 |
Get IRDye® secondary antibodies
Example of normalization against housekeeping gene
Do normalization with an internal control in the same blot, without the inconveniences of stripping and reprobing again.
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 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) |