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).
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