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IRDye® conjugated secondary antibodies

Related

  • Secondary antibodies
    • IRDye® conjugated
      • IRDye® 800CW
        • IRDye® 680RD
        • HRP conjugated
          • AP conjugated
            • Alexa Fluor® conjugated
                • Western blot tools
                  • Fluorescent WB guide
                    • Fluorescent WB Protocol
                        • Fluorescence
                          • Fluorescence guide
                            • Direct vs indirect IF

                              ​​​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
                               secondaries

                              IRDye® conjugated secondaries


                              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

                              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

                              ​
                              Get IRDye® secondary antibodies


                              >>Read next: Hints and tips for successful fluorescent western blot


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