Transition from Chemiluminescent to Fluorescent Western Blots

Dilute ladder

Some pre-stained protein ladders fluoresce strongly and can interfere with detection of proteins expressed at a low level. Try diluting the ladder 1 to 10 before loading.

Optimize sample concentration

As with any new assay, quality results are dependent on first optimizing your assay for the samples to be used. Sample load volume may need to be adjusted for the best possible results. When first switching from chemiluminescent to fluorescent Westerns, try a slightly higher sample load volume to confirm that your assay is working. Once you have a positive result, you can lower the sample load volume and push for more sensitive results.

Be wary of loading dye

Many loading dyes can fluoresce. To prevent this from interfering with the signal on a fluorescent blot, run the dye front off the gel or cut it off before transfer.

PVDF membranes

For the best signal-to-noise ratio, use a membrane with minimal autofluorescence such as Azure’s low fluorescent PVDF membranes.

Membrane handling

Any contamination to the membrane will be obvious in a fluorescent Western blot. Always use forceps when handling membranes.

Probing the membrane

Blocking

As with chemiluminescent blots, it is important to completely block the membrane to avoid nonspecific binding of antibodies and to reduce background. Azure Protein Free Blocking Buffer and Azure Fluorescent Blot Blocking Buffer are formulated to not only reduce background, but also to stabilize the fluorescent dyes of AzureSpectra secondary antibodies for enhanced signal detection.

Optimize antibodies

Antibody concentration may need to be optimized and adjusted for the best possible results. When first switching from chemiluminescent to fluorescent Westerns, use the manufacturer’s recommendations for antibody dilutions.

Incubation trays

Fluorescent secondaries can become quenched when exposed to bright light for long periods of time. When incubating your sample with light sensitive antibodies, cover your blot with an opaque material (such as with Azure’s opaque incubation trays) to protect from quenching.

Stringent wash

Because the fluorescent dyes can adhere to the membrane, washing is extremely important to reduce background. Make sure to use a stringent wash, especially when working with near-infrared fluorophores. Azure Fluorescent Blot Wash Buffer is specially formulated for use with fluorescent Western blots.

Wash volume

The volume and duration of washing is also important to rid the membrane of any free dye and antibody. We recommend two quick rinses in 25mL wash buffer followed by three 5 minute washes in 25mL wash buffer.

Final rinse

Any detergent in the wash buffer can also fluoresce, so rinsing in PBS or TBS after the final washing step is essential to lower background signal.

Reduce background contamination

When many people use the same imager, the insides and trays can become contaminated. Background Quenching Sheets absorb background fluorescence to improve signal-to-noise ratios.

A demo kit is a great way to try out fluorescent Western blotting in your own lab with your own samples and primary antibodies. It contains everything you need to perform single color fluorescent Western blotting:

• PVDF Membrane
• Fluorescent Blot Wash Buffer
• Fluorescent Blot Blocking Buffer
• Secondary Antibody
• 2 Quenching Sheets