Precautions for fluorescence western blot

Precautions for fluorescence western blot

Summary

With the popularization of fluorescence detection, many researchers are considering shifting the detection method of western blot from chemiluminescence to multiple fluorescence. There are multiple driving forces behind this trend. The most important thing is that fluorescence detection can realize multiple western blot analysis, which can detect several target proteins at a time, without stripping and rehybridizing. Other benefits of fluorescence are wider dynamic range and better signal stability.

To make the conversion process easier, scientists at Bio-Rad have brought us some tips for fluorescent western blot.

Fluorescent blotting tips

• The antibody concentration should be optimized by incubating the membrane in several different dilutions of antibody. Select the dilution with the highest signal-to-noise ratio.

• When switching from chemiluminescence to fluorescence detection, the concentration of primary antibody should be increased; usually it is increased by 2-5 times. The secondary antibody concentration may also need to be optimized; a 1: 5,000 dilution is a good starting point. When using specific antibodies, refer to the manufacturer's recommendations. • In order to maximize the signal-to-noise ratio, a membrane with low autofluorescence should be used, such as Immun-Blot® low fluorescence (LF) PVDF membrane.

• Many blocking solutions have been successfully used for fluorescence detection. We recommend using 0.5-5% casein, up to 5% skimmed milk powder, or up to 3% BSA, dissolved in TTBS.

• Particles in the buffer may stay on the membrane and cause fluorescence artifacts. Use only high-quality reagents and filter sterilize all buffers.

• Use blunt forceps to operate from the edge. Avoid scratching or wrinkling the film, which can cause artifacts in fluorescence detection.

• Use a pencil to mark the membrane because many inks fluoresce.

• Bromophenol blue fluoresces. Make sure that the dye is separated from the sample, cut off the portion of the gel that contains the dye, or omit the bromophenol blue in the sample buffer.

• No need for immunoassays in the dark; normal room lighting will not photobleach fluorescently labeled antibodies. However, stock solutions of fluorescently labeled antibodies should be kept in a dark place.

• When handling the membrane, use powder-free gloves to avoid artifacts or fingerprints on the membrane.

Tips for multiple analysis

• Use primary antibodies from different hosts (such as mice and rabbits). Antibodies from two closely related species (such as rats and mice) usually cause cross-reactions, even if the antibodies have been cross-adsorbed.

• Use cross-adsorbed secondary antibodies to avoid cross-reactions.

• Use different fluorophore conjugates in the spectrum to avoid cross-channel fluorescence.

• Before detecting multiple targets simultaneously, optimize the detection of each target individually. Since some primary antibodies are not very specific, multiple bands will be generated on the membrane, so the single target detection before multiple experiments will help determine the band pattern of each antibody.

• Most films will show a higher background under shorter wavelength excitation light. Remember, the strongest target is detected in the blue channel, the medium target is detected in the green channel, and the red channel is reserved to detect the weakest target.