Practical Tips for Fluorescent Western Blots

Here are some practical tips from Advansta for every step of your fluorescent Western blot.

• Make sure samples are completely lysed to maximize recovery of proteins.
• Keep samples cold to prevent protein degradation.
• Use protease inhibitors in lysis buffer to prevent degradation of the sample.
• Determine the protein concentration of each sample to calculate the amount of sample to load on the gel.
• When freezing samples, make small aliquots to prevent multiple freeze-thaws.

• Make sure the gel has no imperfections and is properly set before using.
• Heat samples prior to loading to break secondary structures. Use a reducing agent when required.
• Make sure the well size is appropriate for the sample volume to prevent carryover of samples.
• Do not overload the lane with too much protein; it will cause distortion of the lane.
• If using fluorescent molecular weight markers, skip a lane before loading test samples in case the signal from the markers is intense.
• Run the gel at the correct voltage: running the gel at a voltage that is too high can cause fuzzy bands or “smiling” bands.
• Avoid the use of bromophenol blue which autofluoresces and can cause high background.

• Use a low autofluorescence PVDF membrane to prevent high background.
• Handle the membrane with gloved hands to prevent marks on the membrane.
• Use “powder-free” gloves. Particles in powdered gloves can stick to membranes and cause high background.
• Wash gloved hands before handling membranes and minimize direct contact with working areas. Even non-powdered gloves can contain a tiny amount of residual process chemicals that stick to membranes and cause background or other artifacts.
• Wash all transfer equipment including transfer pads to reduce background.
• Make sure PVDF membrane is pre-wetted with methanol.
• Remove all air bubbles when setting up the transfer sandwich to prevent incomplete transfer or fuzzy bands.
• Optimize the transfer conditions.
  • Alter the concentration of SDS and methanol in transfer buffer to increase transfer efficiency.
  • Decrease transfer time for smaller proteins, increase transfer time for larger proteins.
  • Use a high performance transfer buffer such as FLASHBlot to enhance transfer efficiency and increase protein retention on the membrane.
• Chill transfer setup if necessary to prevent overheating.
• Use a fluorescent stain to monitor transfer.
• Use a pencil to mark the blot as some inks autofluoresce.

• Prepare blocking buffer fresh just before use to prevent contamination and high background.
• Filter the blocking solution to prevent clumps that will stick to the membrane causing speckles and blotches.
• Completely solubilize detergent in the blocking buffer prior to use to prevent high background.
• Optimize the blocking agent to reduce background and increase signal.
  • Be aware that excessive blocking can mask some antigens. Use 1% milk rather than 5%.
  • Try different blocking agents to reduce background.
    • Primary antibodies can recognize components of blocking agents causing high background.
    • Some detection reagents cross-react with blocking solutions.
    • Try a blocking agent optimized for fluorescent detection, such as AdvanBlock-PF.
• Use a TBS-based buffer system when detecting phospho-proteins.
• Do not use milk-based blockers or casein if you plan to use any biotinylated antibody and streptavidin detectors. Milk contains biotin and will cause high background when combined with these reagents.

• Keep all containers and solutions free of dirt to prevent background on blot.
• Cover containers when incubating or washing blot to prevent particulates from falling onto the blot.
• Use a shaker to prevent uneven distribution of buffers.
• Increase wash time to decrease high background.
• Decrease wash time to increase signal.
• Photobleaching is not a concern during incubations and washes – perform these steps on the bench.

• Store fluorescent antibodies in a light-protected box.
• Make sure the primary antibody can detect the protein of interest under the conditions used (i.e. some antibodies only recognize tertiary structures in native proteins).
• Titrate antibodies to yield highest signal-to-noise ratio. Fluorescent Westerns often require different antibody concentrations than chemiluminescent Westerns.
  • Primary antibodies may need to be increased 2-5 fold.
  • Secondary antibodies may also need to be increased (1:5000 recommended starting dilution.)
• To decrease general background, dilute the secondary antibody further.
• To decrease multiple bands that are non-specific, dilute the primary antibody further.
• Use positive and negative control samples to make sure the antibody is detecting the right target.
• Centrifuge antibody aliquots prior to removing sample to avoid aggregates that can cause patches or splotches on the blot.
• Optimize the primary antibody incubation time. While a 1-2 hour incubation at room temperature works well for most antibodies, some antibodies require an overnight incubation at 4°C for optimum performance.

• Use multiplexing to detect multiple proteins simultaneously:
  • Perform in-lane normalization controls by simultaneously detecting the loading control
  • Avoid stripping and reprobing or running multiple blots.
  • Detect proteins of similar molecular weight.
  • Quantify post-translational modifications.
• Use primary antibodies made in different species to prevent cross reactivity.
• Use secondary antibodies that are highly cross-adsorbed.
• Use a a kit designed for multiplexing.
• Use fluorophores with optically distinct spectra to avoid spectral overlap.
• Always optimize detection of each target separately prior to simultaneous detection.
• Detect strong targets in the blue or green channel and weaker targets in the red channel to enhance visualization.

• Make sure detection equipment is compatible with the fluorescent dyes for both excitation and emission wavelengths.
• Clean imaging surface regularly to prevent contamination.
• Protect archived blots from light.

Photo courtesy of Danel Solabarrieta.