One of the most common questions when troubleshooting problematic Western blots is, “Why is the background so high?”
High or uneven background doesn’t only look bad- it also interferes with data analysis, making it difficult to quantify bands or compare bands between samples. There are several things you can do to reduce background and increase the signal-to-noise ratio on your blots. Read on for steps to help you achieve high-quality data and publication-worthy images!
5 Steps to Reducing Background in Western Blots
STEP 1: Use clean, fresh buffers
Make sure your blotting and wash buffers are made fresh. You may want to filter them to remove dust or particulates that may be deposited on your blot and interact with your antibodies or other components of the blotting protocol.
STEP 2: Use the correct blocking agent
Make sure you select a blocking agent that doesn’t interact with your antibody or block your epitope! Commonly used protein-based blocking agents can be problematic in specific situations, particularly with anti-phosphoprotein antibodies. Unsure which blocking buffer to use? Click below for a free sample.
STEP 3: Don’t skimp on the wash steps!
Make sure you use sufficient wash buffer, wash for a long enough time, and agitate the membrane well during wash steps. Any non-specifically bound antibody left on the blot is going to contribute to high background. You may also consider adding additional detergent or changing the detergent in the wash buffer.
STEP 4: Find the best exposure time for your chosen detection method
When over-exposed, any blot can appear as solid background. Ideally, the signal from specific bands is much stronger than any background noise and a short exposure will pick up only the specific signal. If using film, be prepared to expose the blot multiple times to different pieces of film for increasing periods of time to find the optimal exposure. Imaging using an imager like the Azure 600, or another digital imager with a CCD camera makes capturing multiple exposure times even easier.
If you’re using an ECL detection system, use Radiance ECL, a detection reagent with a stable, long-lasting signal, so exposure times are predictable and reproducible. Using Radiance helps ensure the signal doesn’t decay so rapidly that you cannot conduct multiple exposures.
STEP 5: Optimize your antibody concentrations
This is a situation where some initial work up front can save you a lot of time down the line. Using too much antibody can increase the amount of antibody that binds non-specifically to the membrane. Start with the antibody dilution recommended by the antibody provider.
- If background is high, dilute the antibody more, increasing the incubation time if necessary.
- Incubating at 4 °C can also help reduce non-specific binding.
Quick Tips to Keep in Mind for Fluorescent Western Blots
With these tips, you’re on your way to reducing the background and getting clean, clear Western blots. If you still have questions, fill out the form on the right and one of our experts will reach out to assist.
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Shop Reagents for Western Blotting
Azure Fluorescent Blot Blocking Buffer
AzureSpectra Fluorescent Secondary Antibodies
Azure Fluorescent Blot Washing Buffer
Azure Protein-free Blot Blocking Buffer
Azure Chemi Blot Blocking Buffer