Two colors. Two proteins. One western.

Power your experiments with the superior fluorescence technology of Alexa Fluor® dye–labeled antibodies for the consistency and high quality you need. Using Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies, you can generate multicolored western blots that can be imaged on standard near-IR fluorescence scanners such as the LI-COR® Odyssey® Imaging System and the Kodak® image station.

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Advantages of multicolor western detection

Multicolored western analysis with fluorescent dye conjugates enables the simultaneous evaluation of multiple proteins on the same blot, even if the proteins co-migrate. Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies and streptavidin conjugates are ideal for fast and accurate multicolored western detection. Advantages of using multiplexing westerns include:

Saves time—

  • Requires only two antibody incubation steps
  • Eliminates the need for stripping and re-processing your blot

Saves sample—

  • One gel, one blot, one lane per sample

Simplifies data comparison—

  • Diminishes the introduction of artifacts into your experiment seen with multiple blots
  • Allows direct comparison of your samples on the same blot

Sample normalization

GAPDH is a housekeeping gene commonly used for normalization when performing expression analysis. GLUT4 is a glucose transporter found in adipose and muscle tissue. Figure 1 illustrates the use of Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies for simultaneous detection of GAPDH and GLUT4 by western blot analysis.

 Alexa Fluor® 680 and 790 Dyes in Western Detection Normalization 
   
 Figure 1. Simultaneous detection of GLUT4 and GAPDH. GLUT4 and GAPDH were detected simultaneously in 3T3-L1 adipocyte lysates using mouse anti-GAPDH and rabbit anti-GLUT4 with Alexa Fluor® 680 goat anti–mouse IgG and Alexa Fluor® 790 goat anti–rabbit IgG. Single-color images were merged to visualize both proteins. GAPDH bands are shown in red, and GLUT4 bands are pseudocolored green. Alexa Fluor® antibody conjugates Alexa Fluor® 680 and Alexa Fluor® 790 secondary antibodies. 

Phosphoprotein analysis

Total and phosphoprotein content can be detected in the same sample, with no blot stripping and only two antibody incubation steps required. Figure 2 shows the simultaneous detection of total AKT (protein kinase B) and phosphoprotein by western blot analysis.

 Alexa Fluor® 680 and 790 Dyes in Western Detection Phosphoprotein Studies 
   
 Figure 2. Induction of AKT (protein kinase B) phosphorylation in response to insulin treatment. Lysates of insulin-treated and untreated adipocytes were electrophoresed and transferred to nitrocellulose. Blots were incubated with rabbit anti-AKT and mouse anti–phospho AKT primary antibodies simultaneously, then with Alexa Fluor® 680 goat anti–mouse IgG and Alexa Fluor® 790 goat anti–rabbit IgG simultaneously. Total AKT protein bands are shown in red, and phosphoprotein AKT bands are shown in green. Single-color images were overlaid; yellow bands indicate insulin-treated samples where phosphorylation of AKT has occurred. 

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FOR RESEARCH USE ONLY.  NOT INTENDED FOR ANY ANIMAL OR HUMAN THERAPEUTIC OR DIAGNOSTIC USE.