I previously used Ultrahyb buffer for my northern blotting and was able to remove non-specific bands with high and ultra-high stringency washes. Now, I am performing the same northern blotting but using PerfectHyb buffer. With the new buffer, I am observing many more non-specific bands. The band I am most interested to remove (which is about the same size of my band of interest) even shows a much stronger signal with perfectHyb, but even with a high or ultra high stringency wash, the non-specific bands persist while my bands of interest are also diminished. Could you provide any suggestions on how to address this issue without changing the probe or buffer?
It seems like you're encountering a significant increase in non-specific binding when using PerfectHyb buffer compared to Ultrahyb. This could be due to several factors:
Potential Solutions:
Experimental Tips:
- Create a Wash Buffer Gradient: Prepare a series of wash buffers with increasing stringency (e.g., varying temperature and salt concentration). Test these buffers to identify the optimal conditions for your specific experiment.
- Consider a Pre-Hybridization Step: Pre-hybridizing the membrane with a solution containing blocking agents and high salt concentration can reduce background noise.
- Optimize Membrane Preparation: Ensure that the membrane is properly activated and treated to minimize non-specific binding.
- Visualize the Membrane Post-Hybridization: Before washing, visualize the membrane to assess the level of non-specific binding. This can help you determine the appropriate wash conditions.
By systematically adjusting these parameters, you should be able to reduce non-specific binding and improve the signal-to-noise ratio of your northern blots. If you continue to encounter issues, consulting with experienced researchers or technical support from the buffer manufacturer can provide additional insights.
Ali Pirnejad
Thank you for your suggestions, Actually, I am using the same hybridization buffer for the pre-hybridization step, but I'm unsure if this is sufficient or if I should consider using a different solution. I wasn’t aware that this step plays an important role in blocking non-specific bands. Well, I know that increasing the temperature in pre-hyb step affects probe specificity, but I didn’t realize the pre-hybridization step is critical for reducing non-specific bands.
Additionally, I perform a blocking step after the hybridization and washing steps, using the blocking buffer from LicorBio. Should I make my own blocking buffer? then I can change the concentration based on my need.
I didn’t get the point about membrane preparation. I don’t know how should I active the membrane. I am using the positive charged nylon membrane.
I like the idea of visualizing the membrane post-hybridization. Does it affect the signals or no?
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