Some Suggestions for Fluorescent Western Blot

Fluorescent western blotting is a technique for directly detecting protein expression levels by fluorescently labeled detection antibodies. This method is becoming increasingly popular due to recent advances in digital imaging techniques and fluorescent western blotting reagents.

Try to carry out fluorescent western blotting in your lab? You can use the following tips to optimize your protocol and enhance your Western Bloting experience.

1. Increase sensitivity

Use high-sensitivity labels. Quantum dot nanosphere fluorescent secondary antibodies will help achieve higher sensitivity.

If the target protein cannot be detected, try drying the membrane before developing it. Drying may increase the signal intensity by 4-10 times.

Reduce the concentration of sample, primary and secondary antibodies, which minimizes non-specific interference in the experimental sample.

Switching to a less aggressive blocker. It can increase the signal intensity, but this also increase non-specific signals. Examples include diluting casein to 0.5x or using fish or bovine serum albumin (BSA) blockers.

2. Reduce non-specific adsorption

Using casein as a sealer minimizes non-specific binding.

If the signal is strong and there is a lot of non-specific binding, dilute the primary and secondary antibody concentrations.

3. Reduce background fluorescence

Using long-wavelength fluorescent antibodies (e.g., 620 nm) can significantly reduce the autofluorescence of the membrane.

Using short wavelength fluorescent antibodies (e.g. 520nm) to detect abundant targets, such as internal reference proteins (GAPDH).

Use low fluorescence (LF)-PVDF membranes as they minimize the background level (compared to conventional PVDF membranes, and some extent, nitrocellulose membranes).

Avoid using colored ladder, use non-pre-stained or blue pre-stained ladder as much as possible, and keep ladder loading to a minimum.