Why switch from chemiluminescence detection to fluorescence detection

The Western blot imaging method depends on the label of the secondary antibody. The commonly used methods are chemiluminescence based on enzymatic reaction, visible fluorescence method based on fluorescent dye and infrared fluorescence method. How should we choose?
The most suitable Western blot method depends on what information you expect, that is, depending on the purpose of your experiment, Xiaobian compares the advantages and disadvantages of the existing methods, and shares their own experiences, hoping to help you choose the best Experimental method.

Chemiluminescence method

Chemiluminescence western blot is a relatively easy and highly sensitive detection method with sensitivity up to fg.
Suitable for research: proteins with significant molecular weight differences that can be easily separated by electrophoresis.

> Detecting a single protein
> Determine if the protein is present
> Detect antibody response
> Protein purity analysis
> Detect low abundance protein

Fluorescence detection method

Fluorescent dyes are classified into visible fluorescent dyes and infrared fluorescent dyes, so that fluorescent western blot detection is divided into visible fluorescence detection and infrared fluorescence detection. At present, it can be seen that the fluorescence detection can be up to three channels, and three different proteins can be detected simultaneously. The near-infrared detection can be up to two channels, and the laser source must be selected to be excited. The closer to the near-infrared dye, the maximum emission wavelength is 680 nm and 800 nm. Anti-signal, improve the sensitivity of detection.
Fluorescent Western blot was detected by fluorescent dye-labeled secondary antibody, and the target protein concentration on the membrane was linear with the intensity of visible fluorescent signal, realizing reliable and quantitative analysis. Fluorescent dyes have different emission spectra, so multiple detections can be performed on one blot. The post-translationally modified protein can be analyzed by performing sample quality control without stripping and re-incubation.
So when you carry out the following laboratory, choose the fluorescence detection method, properly:

> Simultaneous detection of multiple proteins
> Research on post-translational modified proteins
> Loading quality control of the same imprinted membrane
> Accurate quantitative western blot experiments

Journal journal requirements:

For example, "Image Integrity and Standards" in nature encourages the detection of internal and target proteins on a single blotting membrane, using reasonable standardization methods: for example, whole protein normalization.



Chemiluminescence is an enzymatic reaction with a narrow dynamic range. It can only be qualitatively analyzed. Only one signal can be detected at a time. If multiple signals are to be detected, it needs to be stripped and re-incubated. If film imaging is used, it is necessary to repeatedly explore the exposure time and develop. Fixing equipment and equipment maintenance, fluorescence detection can meet the requirements of journals and magazines, can perform multiple signal detection at one time, consistent reaction conditions, accurate quantification, especially near-infrared fluorescence detection method, with low background, high signal-to-noise ratio, is now Western blot fluorescence quantitative gold standard.