To avoid any chance of potential photobleaching, protect the membrane from the light during incubation and wash steps with aluminum foil.Ěvoid membrane stripping if the goal of the experiment is accurate quantification or multiplexing.Therefore, we suggest using high-quality reagents, allowing suitable time for all components to fully dissolve, and filter sterilize all buffers. ěe wary of anything that can settle on the membrane and create fluorescent artifacts:ġ) Pen marks can have some autofluorescence, so use a pencil.Ģ) Handle the membrane with care use blunt forceps and avoid scratching/creasing to prevent fluorescent artifacts.ģ) Undissolved particles within buffers, such as milk powder in blocking buffer, can potentially settle on the membrane and create fluorescent artifacts.Many vendors supply fluorescence-optimized western blot blocking buffer in both PBS- and TBS-based formats. Like membranes, blocking buffers can autofluoresce thereby giving a higher background.If PVDF membranes are required, there are low fluorescence PVDF membranes available from some vendors.

Nitrocellulose membranes are considered to be the best option for low background compared to traditional PVDF membranes.

Membranes can autofluoresce, which may result in high background.

Steps and reagents to pay attention to in your protocol

Optimize conditions for individual antibody pairs (primary and conjugated secondary) separately, before attempting multi-color analysis.

Test several dilutions and select the one that yields the highest signal-to-background ratio.

ğind the optimal antibody concentration by individually titrating your primary and secondary antibodies.

Optimize the primary and secondary antibody concentrations to get the best signal-to-noise ratio: However, as with ECL western blot, expect a weaker signal than when using a secondary antibody for detection.

Primary antibodies conjugated to the correct fluorescent dye can also be used.

Use secondary antibodies that are highly cross-adsorbed to minimize cross-species reactivity.

When multiplexing, ensure that each primary antibody used is from a different species or isotype.

ğor clear and bright bands, use antibodies that have been properly validated in western blot.So, what do you need to keep in mind when making the switch from ECL to fluorescence? Below are a few useful tips on how to get the best possible results out of your next fluorescent western blot experiment. In this video, you’ll learn about the difference between the ECL and fluorescent detection systems as well as their main advantages and disadvantages. Each has advantages and disadvantages, which depend on your needs and equipment available in your lab. The most common detection methods for western blot include chemiluminescent (ECL) and fluorescent detection. Finally, we’ll show you how to solve your fluorescent western blot problems with some troubleshooting tips.Ĥ.1 Switching to fluorescent western blotting We then go through a detailed protocol for fluorescent western blot. First, we cover the advantages of fluorescent western blot and share useful tips on how to make the switch from chemiluminescence to fluorescence. In Part 4 of our series on western blot, we explain how to perform fluorescent western blotting. We’ll guide you through western blot basics and essential protocols before moving on to optimization, troubleshooting, and more advanced techniques. Welcome to our training series on western blot.

Dot blot vs western blot