10 Top Tips for Converting Western Blots from Chemiluminescence to Multiplex Fluorescence

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Chemiluminescence is the most familiar method of detection for Western blotting and offers great sensitivity; however, many scientific questions and experimental designs require the additional information provided by fluorescent Western blotting. Fluorescent detection provides precise quantitation and visualization of similarly sized proteins within the same sample. If you want to convert from a chemiluminescent Western blot protocol to a multiplex fluorescent Western blot protocol, you’ll need to carefully consider and optimize your current protocol for the best results. To ensure this change is done effectively, first review this standard chemiluminescent Western blot protocol before beginning to pre-optimize a fluorescent protocol.

In this blog post, we share our ten top tips to help you successfully convert from chemiluminescence to multiplex fluorescent Western blots (shown below).

Digital image of 3-color fluorescent multiplexed Western blot
Digital image of 3-color fluorescent multiplexed Western blot using Azure Biosytems c600 imager. Lanes (from left to right) loaded with 1, 2, 5, 10, 20 µg HeLa cell lysate. Probed for tubulin (top), beta actin (middle) and GAPDH (bottom).

Table of Contents

Tip 1: Dilute your ladder

With a fluorescent protocol, only a small amount of ladder is needed. Many brands of the molecular weight ladder need to be diluted 1:10 in loading buffer. This will result in a very faint ladder on the blot, which may cause alarm at first. Know that this is expected and required for high quality fluorescent Western images.

Tip 2: Get rid of the dye front

The dye front can cause background fluorescence. Because of this, be sure to either let the dye front run off the gel during electrophoresis, or cut the dye front off the gel before transferring to the membrane.

Tip 3: Optimize transfer conditions

The type of transfer used can affect the outcome of a multiplex fluorescent blot. Using a wet transfer is ideal as it allows for optimization and quantitative analysis  due to their high customizability. To ensure complete transfer of a broad range of proteins, the time, temperature, voltage, and buffer can be varied based on the protein of interest. With the semi-dry transfer method, the transfer time cannot be extended to allow for more protein transfer and has the potential to dry out due to the limited amount of buffer used. This means that with a semi-dry transfer, proteins at either extreme of the size range will have difficulty transferring.

  • Quick Tip: Pre-soak the low fluorescence PVDF membrane in methanol to activate the membrane for transfer.

PVDF membranes are best for fluorescent Western blots. To ensure background fluorescence is not an issue, use low fluorescence PVDF membranes. These ready-to-use PVDF membranes are conveniently available in three pre-cut sizes for you to choose from.

Preparing to transfer an SDS-PAGE gel to a membrane for a Western blot.
Preparing to transfer an SDS-PAGE gel to a membrane for a Western blot using ready-to-use PVDF membranes from Azure Biosystems.

Once the transfer is complete, do not write with ink or pencil on the membrane; the graphite and ink will bleed and autofluoresce. After the transfer, do not expose the membrane to any container that has ever been exposed to Coomassie stain. Only use clean, dedicated Western blot trays. 

Tip 4: Take the guesswork out of blocking

Blocking fluorescent Westerns can be tricky. Initially, using a dedicated fluorescent blot blocking buffer, like this one, makes the transition to fluorescence from chemiluminescence much simpler and easier.  We highly recommend using Azure Fluorescent Blot Blocking Buffer, as it stabilizes fluorescent signals and comes ready to use. Once you have optimized other parts of your protocol, then you can test milk and other blocking buffers.

Read more: Getting Rid of the Noise: Western Blot Blocking

Tip 5: Skip Ponceau staining

Ponceau stain can increase background fluorescence. Since our end goal is a beautiful fluorescent Western blot, avoid any reagents because using them could increase background fluorescence.

  • Quick Tip: After blocking, never do a Ponceau stain on the membrane.

Tip 6: Ensure the primary antibodies are optimized

Before transitioning to a multiplex fluorescent protocol, evaluate how your current Western results look. Which proteins do you intend to multiplex together? Are the current antibodies you are using for the different proteins resulting in clean, sharp bands with low background? If not, optimizing the primary antibodies is necessary.

Direct antibody labelling allows the use of antibodies from the same species or of the same isotype and reduces the potential for cross reactivity.

Read more: Antibody Labeling in the Lab

Tip 7: Use the right wash buffer

The differences in image quality caused by wash buffer choice alone can be dramatic. Using a buffer that is specific for fluorescent Westerns is ideal here. Check out this fluorescent wash buffer. It’s perfectly optimized for use with fluorescent secondary antibodies.

See the difference using a fluorescent wash buffer can make for yourself by preparing two blots in parallel using different types of buffer. We also show this in the comparison images below.

Comparison of using fluorescent Washing Buffer vs. insufficient wash
Azure Fluorescent Blot Washing Buffer is optimized for use with both visible and near-infrared fluorescent Western blots. It is specially formulated to reduce non-specific binding of fluorescent secondary antibodies to produce clean blots with high signal-to-noise ratios.

If you don’t have this premade buffer readily available, using TBST with 0.1% Tween20 is an acceptable substitute.

  • Quick Tip: The final wash step should be performed with either TBS or PBS with no detergent. After the final wash, dry the membrane by air drying on a quenching sheet in a dark cabinet or drawer. Dipping the membrane in methanol can help it dry more quickly.

Tip 8: Optimize your proteins for fluorescent detection

Typically, proteins that require less than 3 minute exposures with film can be detected reasonably well with fluorescence. If you are currently using a strong ECL substrate with greater than 5 minute exposures to detect the protein of interest, you will likely need to optimize your protein fluorescence detection to get optimal results.

Optimizing your proteins is easy to do with fluorescent Western blotting kits, such as this one. It is a full kit with everything you need to perform ten two-color fluorescent Western blots.

AzureSpectra Fluorescent Western Blotting Kits with Fluorescent Block Each kit contains enough materials for 10 fluorescent Western blots: 9x7cm Low Fluorescence PVDF Membranes, 10 membranes 10x Fluorescent Blot Washing Buffer, 250mL 1x Fluorescent Blot Blocking Buffer, 300mL Background Quenching Sheets, 2 sheets 2 Fluorescent Secondary Antibodies, 40μL each
Fluorescent Western Blotting Kit from Azure contains enough materials and reagents for 10 fluorescent Western blots.

Tip 9: Protect from light

Once the fluorescent secondary antibodies have been added to the membrane, protecting the membrane from light is crucial. During washes and imaging, keep exposure to light to an absolute minimum to ensure the strongest fluorescent signal.

AzureSpectra secondary antibodies are labeled with fluorophores that emit light in visible and near-infrared wavelengths. AzureSpectra 490, 550, 650, 700 and 800 secondary antibodies offer unparalleled sensitivity and performance for immunoblotting applications when used in conjunction with Azure Imaging SystemsDue to low background autofluorescence in the near-infrared region, AzureSpectra 700 and 800 secondary antibodies can produce higher signal-to-noise ratios.

Tip 10: Choose the right fluorescent channel

A good rule is to detect the most abundant proteins with the IR700 channel You can use the IR800 channel to detect less abundant proteins. If both proteins being evaluated require less than a one-minute exposure on film, either channel may be used to detect either protein.

Choosing the correct fluorescent channel on the Azure 600 Imager.
The Azure 600 is the only system that offers two channel, laser-based IR and chemiluminescent detection, with the speed and sensitivity of film. It has the ability to image visible fluorescent dyes, standard EtBr and protein gels, and laser excitation for quantitative Western blot imaging in the NIR.

The Azure 600 is an imager that offers laser technology with two IR detection channels, enabling you to image more than one protein in an assay. It provides accurate and fast chemiluminescent detection, as well as the sensitivity, dynamic range, and linearity needed for quantitative blot analysis.

Use an imager with multiplex capabilities

Fluorescent Western blots are visualized using an imager, like the Azure Sapphire FLUsing an imager with multiple channels bypasses the need to strip and re-probe membranes. Newer imaging systems have sophisticated detectors with the ability to exhibit a broader dynamic range than film and therefore avoid signal saturation issues.

Azure Sapphire FL Biomolecular Imager with lid open
The Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types, including arrays, microarrays, Western blots, tissue slides, and small animals.

The Sapphire FL can detect up to four proteins on the same blot and overlapping bands can be quantified. This saves you money, reagents, and reduces your total imaging time.

We are committed to supporting your research and ensuring a seamless transition to multiplex fluorescence in your Western blotting experiments. By following these best practices and keeping these ten fluorescent multiplex tips in mind, we hope you’ll find that converting your existing chemiluminescent Western blot protocol into a multiplex fluorescent Western blot protocol can be both simple and successful. If you encounter any difficulties or have questions, please reach out to our team for further assistance.

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