Combining WesternDot™ Technology With WesternBreeze® Chemiluminescent Detection

Combining WesternDot™ Technology With WesternBreeze® Chemiluminescent Detection—Multiplexed Western Blot Detection

To visualize more than one protein of interest on a western blot, traditional chemiluminescent or chromogenic detection technology requires preparing multiple blots or sequential stripping and reprobing of a single blot. Simply by integrating WesternDot™ detection technology into your current WesternBreeze® chemiluminescent western blot workflow, two proteins can be visualized simultaneously on the same blot, with no additional solutions or steps.

Combining WesternDot™ Kits With WesternBreeze® Reagents

WesternDot™ 625 western blot kits combine the unique properties of Qdot® 625 nanocrystals with the high-affinity streptavidin–biotin interaction to achieve sensitivity equivalent to chemiluminescence with a simple, no-hassle western protocol. Qdot® 625 nanocrystals have an extremely high extinction coefficient in the UV and blue wavelengths, a high quantum yield, and an emission maximum near 625 nm. These properties allow for subnanogram sensitivity of protein detection using standard detection systems and emission filters. Following a primary antibody incubation, detection relies on a biotinylated goat anti-mouse or goat anti-rabbit secondary antibody followed by a Qdot® 625 streptavidin conjugate.

For WesternDot™ and WesternBreeze® multiplexed detection, a blot is coincubated with both a mouse and a rabbit primary antibody, then with a goat anti-mouse or goat anti-rabbit alkaline phosphatase secondary antibody and the alternate biotinylated secondary antibody, followed by incubation with the streptavidin–Qdot® 625 conjugate. The Qdot® signal can then be imaged before, during, or after incubation with the WesternBreeze® CDP-Star® detection reagent (Figure 1). The remarkable photostability of the Qdot® nanocrystal allows the blots to be dried and re-imaged days and even months later, and the signal is not diminished by the WesternBreeze® enzymatic reaction.

Figure 1. Multiplexed WesternDot™ 625 and WesternBreeze® protein detection. A 2-fold dilution series (10 µg to ~160 ng) of untreated (left side of blots) and wortmannin-treated (right side) Jurkat cell extracts was separated on NuPAGE® Novex® 4–12% Bis-Tris gels and transferred to nitrocellulose membranes using the iBlot® dry blotting system. Blots were coincubated with rabbit anti-AKT and mouse anti-pAKT antibodies and detected with WesternDot™ reagents (left). The same blots were incubated with WesternBreeze® reagents to detect the alternate antigen (right). For Qdot® 625 detection, the membrane was imaged on a FujiFILM LAS-4000 imager with UV epi-illumination, a 605DF40 emission filter, and an exposure time of 20 sec. For CDP-Star® detection, the membrane was imaged without an illumination or emission filter, with an exposure time of 1 min.

Simple, Versatile Western Blot Detection

WesternDot™ reagents can be multiplexed with other chemiluminescent detection reagents (such as our Novex® ECL reagents) or chromogenic detection reagents (such as our WesternBreeze® chromogenic kits).