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