Here are a few useful tips on how to get the best possible results out of your fluorescent western blot experiment.

Choosiantibodies for fluorescent western blot

• To obtain clear and bright bands, use antibodies that have been properly validated in western blot.
• When multiplexing, ensure that each primary antibody used is from a different species or isotype to prevent cross-reactivity.
• Select appropriate internal reference antibodies based on the type of lysate. Keep in mind that certain lysate types, such as plasma, are not compatible with using GAPDH as the loading control.
• Use secondary antibodies that are highly cross-adsorbed to minimize cross-species reactivity.
• Alternatively, primary antibodies conjugated to the correct fluorescent dye can also be used. However, as with ECL western blot, expect a weaker signal than when using a secondary antibody for detection.
• When selecting a loading control, avoid choosing antibodies with molecular weights that overlap with those of your primary antibodies.

Optimizing your antibodies for fluorescent western blot

Optimize the primary and secondary antibody concentrations to get the best signal-to-noise ratio:

• Find the optimal antibody concentration by individually titrating your primary and secondary antibodies. Test several dilutions and select the one that yields the highest signal-to-background ratio.
• Before attempting multi-color analysis, optimize conditions for individual antibody pairs (primary and conjugated secondary) separately.

Steps and reagents to pay attention to in your fluorescent western blot protocol

• Membranes can autofluoresce, which may result in high background. Nitrocellulose membranes are considered to be the best option for low background compared to traditional PVDF membranes. If PVDF membranes are required, there are low-fluorescence PVDF membranes available from some vendors.

• Like membranes, blocking buffers can autofluoresce, thereby giving a higher background. Many vendors supply fluorescence-optimized western blot blocking buffer in both PBS- and TBS-based formats.

• Be wary of anything that can settle on the membrane and create fluorescent artifacts:

  • Pen marks can have some autofluorescence, so use a pencil.
  • Handle the membrane with care; use blunt forceps and avoid scratching/creasing to prevent fluorescent artifacts.
  • Undissolved particles within buffers, such as milk powder in a blocking buffer, can potentially settle on the membrane and create fluorescent artifacts. Therefore, we suggest using high-quality reagents, allowing suitable time for all components to fully dissolve, and filter sterilize all buffers.

• Avoid membrane stripping if the experiment's goal is accurate quantification or multiplexing.

• To avoid any chance of potential photobleaching, protect the membrane from the light during incubation and wash steps with aluminum foil.

• If using bromophenol blue in your loading dye, you should ensure your dye front is cut from the gel before the transfer, as this dye will transfer to the membrane and autofluoresce. If blotting a low molecular weight protein, we recommend using fluorescent western blot loading dyes that lack bromophenol blue and don’t autofluoresce.

Figure 1 shows an example of the fluorescent western blot performed with IRDye® secondary antibodies.

Figure 1. Western blot with goat anti-mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772). All lanes: Anti-p53 antibody [DO-1] - ChIP Grade (ab1101) at 1/1000 dilution (Anti-p53 antibody [DO-1]). Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: p53 knockout HAP1 cell lysate (20 µg) Lane 3: A431 cell lysate (20 µg) Lane 4 Saos-2 cell lysate (20 µg) Secondary in all lanes: Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) at 1/10000 dilution.

This blot was produced using a 4–12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 50 minutes before being transferred onto a nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour before being incubated with ab1101 overnight at 4°C. Antibody binding was detected using the Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 at a 1/10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

The image shows a merged signal (red and green). Green – p53 (ab1101) observed at 53 kDa. Red - GAPDH loading control (ab181602), observed at 37 kDa using as secondary Goat Anti-Rabbit IgG H&L (IRDye® 680RD)- ab216777.

Multiplexing in fluorescent western blotting

Here we discuss the advantages of detecting multiple targets on the same blot at the same time with fluorescent western blotting.

Multiplexing in fluorescent western blotting allows you to:

• Quantify relative protein abundance.
  Compare the abundance of one protein over another. For instance, you can compare the abundance of a phosphorylated form of your protein of interest to the total amount of protein.
• Normalize in the same gel.
  Do normalization of band intensity with an internal control in the same blot without the inconveniences of stripping and reprobing again.

Why is quantification and multiplexing possible with fluorescent WB?

• Fluorescent applications are more quantitative than enzyme-based approaches (eg HRP) , making normalization against an internal control (eg housekeeping gene) easier and more accurate.
• Fluorescent western blotting provides the widest dynamic range , so both low- and high-abundant proteins can be detected in the same experiment.

IRDye® is a registered trademark of LI-COR, Inc.

LI-COR® is a registered trademark of LI-COR, Inc.