At a minimum, a hand-held blue or ultraviolet light source and a high-quality color digital camera can be used to illuminate the blot and capture images of the fluorescent protein bands. Qdot western blot imaging is flexible and can be done at several levels of sophistication. All reagents have been optimized for high sensitivity and low background signal with mouse and rabbit primary antibodies. The kit includes Qdot Conjugates, block and wash buffers and PVDF blotting membranes specially selected for low auto-fluorescence. Each kit is supplied with all the necessary reagents to label ten blots. Although additional colors are available, two colors have been provided to enable flexible multiplex labeling and quantification with existing gel imaging systems. Two western blot kits are available, one containing Qdot 565 nm Goat anti-Mouse Conjugate and Qdot 655 nm Goat anti-Rabbit Conjugate and a second that contains Qdot 605 nm Goat anti-Mouse Conjugate and Qdot 705 nm Goat anti-Rabbit Conjugate. Labeled blots can be stored in a buffer at 4–8 ☌ with minimal loss of signal for imaging at a later date. The procedure requires no additional time other than what is typically done for simple colorimetric detection. The blots are subsequently labeled with Qdot Conjugates and imaged with one of several fluorescence gel imaging systems. Electroblotting from SDS-PAGE gels is performed using routine procedures, and the blots are labeled with rabbit or mouse primary antibodies at concentrations typically used for colorimetric or chemiluminescent detection. Qdot Western Blotting Kits are designed to be a 'plug-and-play' substitute for colorimetric or chemiluminescent detection systems on blots labeled with rabbit and mouse primary antibodies. This high level of brightness and excellent photostability bring the added value of high sensitivity and ruggedness. Qdot nanocrystals are extremely efficient at absorbing light and converting it to a highly stable fluorescent emission, making them up to 50× brighter than conventional organic fluorophores. The narrow emission enables simplified multiplexed labeling, image acquisition and quantification. QDC has developed processes to produce precise size distributions and consequently precise, narrow spectral emissions ranging from 525 to 800 nm. Quantum dots absorb light over a broad spectral range and fluoresce at wavelengths determined by their physical size 1, 2. The fluorescence properties of the quantum dot core are one of the key advantages for its use in western blot applications over currently used detection technology. The nanocrystal is coated with an organic molecular layer that provides water solubility and conjugation sites for biomolecules. Qdot Conjugates are engineered fluorescent materials consisting of a Quantum dot nanocrystal core and shell of CdSe and ZnS, respectively.
These kits use Qdot Secondary Antibody Conjugates to provide a sensitive method of detecting immunolabeled proteins in western blotting applications. QDC has recently introduced two western blotting kits.
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