Western Blot Application Note

Advantages of Western Blots using T2-MP EviFluor Quantum Dots

EviFluors offer major advantages over traditional chemiluminescent and radioactive techniques used in Western blot analysis. In addition to offering the ability to color multiplex it also offers a significant cost savings. Some of the advantages of T2-MP EviFluor based Western Blots include:

Increased Photostability leading to High Sensitivity
Multiplexing capability
Increased Linearity resulting in Improved Quantification of Protein Expression
Cost Savings compared to ECL
Compatibility with existing Imaging Systems

EviFluor Western Blots allow for color multiplexing as well as a broad range of linearity for accurate quantification. Now it is possible to observe the presence and amount of several antigens on the same blot and determine their concentrations, which translates to cost advantages over single color testing.

Enhanced chemiluminescence (ECL) is one of the most universally used blotting techniques with detection limits in the pico-gram range. The non radioactive nature of ECL makes it more attractive than radioisotope techniques which have similar sensitivity. However, even though ECL is fast and sensitive, it lacks the capability of detecting more than one antigen per blot.

Western blot analysis using traditional organic fluorophores have inherent limitations. Low photostability makes it difficult to image over long periods of time, resulting in lower sensitivity. Multiplexing is often difficult with organic dyes due to fact that each dye needs to be excited at different wavelengths requiring multiple excitation sources and complicating the design platform. In addition the broad emission spectra of the dyes can lead to cross talk between channels resulting in spectral overlapping.

Photostability, Broad Absorption, Narrow Emission- A New Way to Multiplex!

The unique quantum dot optical characteristics enable the use of T2-MP EviFluors as superior fluorescent imaging agents for western blotting. Increased photostability provides the benefits of greater signal stability as well as the ability to achieve quantitative analysis. In addition, broad absorption coupled with narrow excitation spectra facilitates color multiplexing in a more simplified system than required with traditional fluorophores. A single, inexpensive UV wavelength excitation source is all that is required to obtain symmetric emission signals from each quantum dot ranging in wavelength from 490 nm to 680 nm.

Western Blot using EviFluors

T2-MP EviFluor Quantum Dots are a ready to use fluorescent probe for Western blotting. These EviFluors are pre-conjugated to commonly used secondary antibodies, which subsequently can be used to target primary antibodies. The EviFluor Western blot procedure takes into account the commonly used membranes, blocking and washing buffers. Imaging of the Western blot can be obtained using simple UV epi-illumination imaging systems with readily obtainable filters.

Sensitivity

Quantum dots? enhanced photostabilty allows for longer integration time of the emission signal, resulting in significant improvements in signal to noise ratios, and their narrow, symmetric emissions facilitate reduced cross talk between signals, resulting in increased sensitivity. Under appropriate storage conditions the photostability of the quantum dots allows the scientist to image blots months after probing.

Protocols

Click here to view our Western Blot Protocol

Click here to view for our In-Gel Western Blot Protocol

Order EviFluor Quantum Dot Conjugates Below

Product Availability
Conjugate	Wavelength	Part Number	Concentration	Price
Goat anti-Mouse	520nm 600nm 620nm 680nm	EF-C11-GMO-0XXX	2.5uM	$289
Goat anti-Rabbit		EF-C11-GRA-0XXX		$289
Goat anti-Rat		EF-C11-GRT-0XXX		$289
Streptavidin		EF-C11-STR-0XXX		$439
Biotin		EF-C11-BT1-0XXX		$439
Specifications