Western blotting is a cornerstone of laboratory research, yet it is notoriously finicky. If your results are looking more like a “blob-y mess” than a clean set of data, you aren’t alone. Improving your assays often comes down to refining a few critical steps in the workflow.
As fellow researchers here at Lectenz Bio, we put our heads together to give you five key reasons your Western blot might be failing and how to fix them.
1. Reagent Specificity and Cross-Reactivity
The foundation of a successful blot is the specificity of your detection reagents. If your antibody or lectin binds to non-target proteins, your results will be uninterpretable.
- The Reality of Antibodies: A 2008 study revealed that only 49% of over 5,000 commercially available antibodies actually recognized their intended target.
- Detection Reagent Cross-Reactivity: This isn’t limited to antibodies; for example, the lectin MAL-I is often used for detecting α2,3 linked-sialic acid but also has a high affinity for sulfated galactose.
2. Inadequate Sample Preparation
High-quality blots require properly prepared starting material.
- Lysis and Boiling: Incomplete lysis or insufficient boiling can prevent proteins from properly denaturing, which leads to laddering on the gel and the final blot.
- Reducing Agents: Ensure you are using the correct concentrations of DTT or BME; errors here will negatively impact how your bands separate during electrophoresis. DTT also oxidizes quickly and should be added immediately prior to sample preparation to ensure maximum efficacy.
3. Suboptimal Blocking
Blocking prevents non-specific binding by coating the “empty” spaces on your membrane with inert proteins like BSA or casein.
- Don’t Be Afraid to “Over”-Block: It is nearly impossible to over-block a membrane. Increasing your blocking reagent concentration or extending incubation times—even overnight—can significantly reduce background noise. This can also be true for primary reagent incubations as well.
- Choose the Right Agent: Casein often blocks more effectively than BSA, but because it is glycosylated, it may interfere with certain glycobiology assays. Also important to note, some detection reagents perform better with different blocking agents.
4. Insufficient Washing
Washing is perhaps the most underrated step in the immunoassay process. It is essential for removing unbound reagents that cause background interference.
- Use Detergents: Incorporating detergents like Tween-20 into your wash buffers helps strip away non-specific binding.
- Take Your Time: For example, if the protocol suggests washing for 5 minutes and repeating 3 times, use the entire time.
5. Selecting the Wrong Membrane
Believe it or not, the way a blot turns out can be directly affected by the type of membrane used. The two commonly used membranes for blots are nitrocellulose and PVDF, which both have their own unique properties. If you’re doing all your blots on PVDF or nitrocellulose and have issues, sometimes just switching the membrane can solve your issues.
- Nitrocellulose: These membranes are porous, which allows the proteins to embed into the membrane and, if the transfer goes too long, even go through the membrane. It is also more brittle than PVDF.
- PVDF: This membrane is non-porous and captures everything transferred to its surface, which can increase background noise. It also needs to be activated with methanol prior to use.
At Lectenz Bio, we create bench-to-bench glycoscience tools aimed at simplifying science for our customers and giving them results they can count on. Part of this means we rigorously test all of our products in a host of assays, carefully formulate binding buffers, and develop validated protocols that work consistently (we also update them). For our reagents, we not only screen them against standards, glycoproteins, and substrates in-house; they are also validated by collaborators in academia and industry in their assays – ensuring we know exactly what to expect from our reagents.