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; this includes precise quantitation and visualization of similarly sized proteins within the same sample. Converting from a chemiluminescent Western blot protocol to a multiplex fluorescent Western blot protocol requires careful consideration and optimization. To ensure the change is done effectively, you must understand your current chemiluminescent Western blot protocol and pre-optimize the fluorescent protocol. In this blog post, we share our ten top tips to help you successfully convert from chemiluminescence to multiplex fluorescent Westerns (example shown below).
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.
PVDF membranes are best for fluorescent Western blots. To ensure background fluorescence is not an issue, use low fluorescence PVDF membranes, like these ready-to-use PVDF membranes from Azure. They’re conveniently available in three pre-cut sizes for you to choose from.
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. We highly recommend using Azure Fluorescent Blot Blocking Buffer, as it stabilizes fluorescent signals and comes ready to use. Initially, using a dedicated fluorescent blot blocking buffer, like this one from Azure Biosystems, makes the transition to fluorescence from chemiluminescence much simpler and easier. Once you have optimized other parts of your protocol, then you can test milk and other blocking buffers.
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.
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. Azure has created a fluorescent wash buffer that’s perfectly optimized for use with fluorescent secondary antibodies. To see the difference a fluorescent wash buffer can make, prepare two blots in parallel each washed with a different type of buffer. We show this in the comparison images below.
If you don’t have this premade buffer readily available, using TBST with 0.1% Tween20 is an acceptable substitute.
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 from Azure. This full kit has everything you need to perform ten two-color 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.
Shop AzureSpectra secondary antibodies, 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 Systems. Due 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 and the less abundant proteins with the IR800 channel. If both proteins being evaluated require less than a 1-minute exposure on film, either channel may be used to detect either protein.
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 FL. Using 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.
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.
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. Azure is committed to supporting your research and ensuring a seamless transition to multiplex fluorescence in your Western blotting experiments.