Fluorescent Western Blotting: Helpful Tips for Optimizing Your Protocol

Fluorescent western blotting is a technique where fluorescently tagged detection antibodies help directly determine the level of protein expression. It is a method that is becoming increasingly popular due to recent advances in digital imaging technology and fluorescent western blotting reagents. Not quite sure about this technique? Read about the reasons to start fluorescent western blotting.

Once you have decided to try fluorescent western blotting for yourself, use these tips to optimize your protocol and enhance your western blotting experience.

Enhance Sensitivity

• Use high-sensitivity labels. StarBright™ Secondary Antibodies will help achieve the highest sensitivity
• If you are unable to detect your target, dry the blot and image again. Drying seems to increase signal intensity 4­–10 fold
• Decrease the amount of sample and primary and secondary antibodies as this can minimize contamination in experimental samples
• Sometimes switching to a less stringent blocker can help increase signal intensity. However, this can also increase non-specific binding. If you go this route, dilute the casein to 0.5x or use a fish-based or bovine serum albumin (BSA) blocker

Minimize Non-Specific Binding

• Use casein as a blocker as it is best in minimizing non-specific binding
• Optimize antibody concentrations by diluting the primary and secondary antibodies if the signal is very strong and there is a lot of non-specific binding

Avoid Crosstalk

• Balance signal intensity in neighboring channels by using the more sensitive label for the weakest signal and vice versa
• Skip a channel. For example, if the spectral crosstalk between StarBright B700 and DyLight 800 is too high and sensitivity is not as important, use DyLight 650 instead of StarBright. Although this will shift the signals apart and improve crosstalk, it will also decrease sensitivity

Decrease Spectral Background

• Using higher wavelength antibodies (for example, between 650 and 800 nm) greatly reduces membrane autofluorescence. Only use lower wavelength antibodies (for example, 550 nm) for very abundant targets such as housekeeping proteins (HKPs)
• Use low fluorescence (LF)-PVDF membranes as they minimize background levels (compared to regular PVDF and, to some degree, nitrocellulose)
• Avoid using dual-color standards since the red/pink dye can interfere with the visualization of proteins detected by antibodies emitting in the rhodamine channel. Use all unstained or all blue standards and do not overload
• Run the dye front off the gel to avoid fluorescence

Get information about Bio-Rad’s new StarBright™ and hFAB™ Fluorescent Antibodies and ChemiDoc™ MP Imaging System, designed for multiplex detection, brighter signal, and a streamlined workflow.

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