Help! Why do my Western blots look terrible?

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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.

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 with a CCD camera makes capturing multiple exposure times even easier.

If using an ECL detection system, find a detection reagent with a stable, long-lasting signal (like Radiance ECL) so exposure times are predictable and reproducible, and 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.

3 Things to Keep in Mind for Fluorescent Western Blots

  • Azure Quick Tip #1: Change your membrane

    Nitrocellulose and some PVDF membranes can autofluoresce. To reduce background from your membrane, use only low-fluorescence PVDF membranes.

  • Azure Quick Tip #2: Remember that wet membranes can also autofluorescence

    Dry the membrane completely before imaging.

  • Azure Quick Tip #3: Control the temperature during the protein transfer step

    Excessive heat during transfer is usually a major source of background in fluorescent Western blotting.

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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