All tags secondary-antibodies IRDye® conjugated secondary antibodies

IRDye® conjugated secondary antibodies


Learn about the advantages of infrared fluorescent western blot using IRDye® conjugated secondary antibodies compared to chemiluminescence (ECL) using HRP secondary antibodies, including multiplex western blot.

    

Western blot fluorescence vs chemiluminescence

Learn the advantages of IRDye® secondary antibodies  that allow you to due 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)

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 how to do normalization against 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 fluorescent western blot for multiplex western blot also allows sensitive and quantitative detection of your protein of interst 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 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


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