Stripping Your Western Blot Membrane: A Recipe for Success

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Introduction to Western Blot Membrane Stripping

Western blot membrane stripping is a valuable technique used to remove both primary and secondary antibodies from proteins on a transfer membrane. Stripping your membrane offers several benefits – it conserves resources, materials, and time. Rather than starting a new blot, creating a stripping buffer and re-probing your membrane can be more cost-effective and scientifically advantageous.

When to Strip Your Membrane

There are instances where stripping can be particularly helpful. It allows for:

Comparing Protein Abundance

Stripping enables you to compare the relative amount of two proteins, providing insights into their levels in your samples.

Detecting Proteins of Similar Molecular Weights

If you need to identify proteins with similar molecular weights, stripping allows you to target specific signals without interference.

Analyzing Loading Control Proteins

Stripping can be utilized to analyze loading control proteins, ensuring accurate normalization and quantification of your target protein.

Confirming Results

Stripping also allows you to confirm your results by using an alternative antibody or repeating the experiment with the same antibody.

Choosing the Right Western Blot Membrane

It’s crucial to consider the type of membrane you use in your experiment if you anticipate the need for stripping. Two common types are Polyvinylidene Difluoride (PVDF) and Nitrocellulose (NC).

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While Nitrocellulose is not recommended for re-probing after use due to the loss of protein signal, PVDF retains the signal effectively, and stripping does not damage the embedded proteins.

How to Strip Your Western Blot Membrane

There are two common methods for stripping your Western blot membrane – mild and harsh stripping protocols. Let’s explore each:

Mild Stripping Protocol: Low pH Glycine Solution

  1. Prepare a solution with:

    • 1.5% (w/v) Glycine
    • 0.1-1% (w/v) SDS
    • 1% (v/v) Tween20 in Deionized water
    • Adjust to pH 2.2

  2. Soak the membrane in the stripping buffer for 20 minutes.

  3. At the 10-minute mark, remove the buffer and apply fresh stripping buffer.

  4. Rinse the membrane twice with Phosphate-Buffered Saline (PBS) for 10 minutes each, followed by two rinses with PBS for 5 minutes each.

The low pH and Glycine solution dissociate bound antibodies, rendering them inactive.

Harsh Stripping Protocol

  1. Prepare a final solution in deionized water containing:

    • 1% SDS
    • 62.5mM Tris HCl, pH 6.8
    • 0.8% beta-mercaptoethanol

  2. Heat the buffer at 50°C and agitate the membrane slightly for 30-45 minutes.

  3. Rinse the membrane thoroughly with running water to remove excess stripping buffer.

  4. Conduct several catch washes using Tris-Buffered Saline with Tween (TBST) to prepare the membrane for blocking.

The harsh stripping protocol denatures the antibodies, ensuring complete removal from the membrane.

Evaluating the Success of Membrane Stripping

To check if the membrane stripping was successful, follow these steps:

  1. Block the membrane with the appropriate blocking agent.

  2. Proceed with the secondary antibody.

  3. If the primary antibody was successfully removed, no signal bands should be detected.

  4. If there are bands, repeat the stripping procedure to ensure all antibodies are removed.

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It’s advisable to test your membrane stripping skills on a negative control before proceeding with your actual Western blot membrane. This will help you gain confidence in your technique and avoid any unexpected surprises.

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Conclusion

Membrane stripping is an invaluable technique in Western blotting, allowing for further data analysis and resource conservation. By following the appropriate stripping protocol and evaluating the success of the procedure, you can confidently re-probe your membrane and obtain reliable results. Choose the right membrane for your experiment and experience the benefits of effective Western blot membrane stripping.

Western Blot Stripping