Western blotting is a key technique for the detection of specific proteins in complex samples. It has a wide range of applications in areas such as diagnostics, protein characterization and expression studies. Researchers want to extract the most information from samples, and stripping and resorption are an important method. Not only do these methods save valuable samples, they also increase experimental efficiency and optimize results.
Why Strip and Reprobe Western Blots?
Optimizing Signal-to-Noise Ratio (S/N Ratio)
In western blotting, the signal corresponds to the band representing the target protein, while noise refers to background signals from non-specific binding. The ultimate goal of any blotting procedure is to maximize the signal-to-noise ratio (S/N ratio) to ensure clear and interpretable results. Stripping and reprobing offer a crucial method for optimizing this ratio, particularly when initial experiments yield ambiguous or low-quality results. By fine-tuning the antibody concentrations and detection conditions through reprobing, researchers can improve the clarity of the desired protein bands without re-running gels.
Sample Conservation
When dealing with rare or valuable protein samples, conserving material is critical. Traditional western blotting requires running multiple gels and transferring proteins to several membranes, which can consume large amounts of sample. Stripping and reprobing reduce the need for multiple runs by allowing the same blot to be reused for probing different proteins or re-optimizing the same target.
Saving Time and Reducing Costs
Running SDS-polyacrylamide gels and transferring proteins onto membranes is a time-consuming process. Stripping and reprobing accelerate the workflow by allowing the same membrane to be used for multiple rounds of detection, significantly reducing time spent on sample preparation. Moreover, this technique cuts costs by saving on consumables such as gels, membranes, antibodies, and substrates.
Efficient Assay Optimization
High-sensitivity chemiluminescent detection methods often require precise antibody optimization to achieve an ideal signal. Stripping and reprobing streamline this process by allowing adjustments in antibody concentrations and reprobing the same membrane, enabling researchers to fine-tune detection conditions without wasting valuable protein samples.
Rapid Confirmation of Results
In cases where experimental results deviate from expectations, reprobing the blot offers a quick way to confirm the presence or absence of specific proteins. Instead of repeating the entire blotting procedure from the beginning, researchers can strip the membrane and reprobe, allowing for the rapid validation of unexpected findings.
Correcting Experimental Errors
The multi-step nature of western blotting introduces numerous opportunities for procedural errors, whether in antibody handling, detection reagent application, or blocking steps. Stripping and reprobing provide an opportunity to correct mistakes without starting the experiment from scratch, saving both time and resources.
How to Strip and Reprobe Western Blot Membranes
The key to successful stripping is to remove the bound primary and secondary antibodies without damaging or removing the immobilized protein of interest. Depending on the specific antibody-antigen interactions and the detection system used, various stripping protocols—ranging from mild to stringent—can be employed.
Membrane Selection
Before considering any stripping protocol, it's crucial to choose the appropriate membrane. Polyvinylidene fluoride (PVDF) membranes are highly recommended due to their high protein-binding capacity (170–200 µg/cm²) and durability. PVDF membranes exhibit better protein retention than nitrocellulose, which makes them ideal for multiple rounds of stripping and reprobing. Moreover, PVDF's hydrophobic nature enhances its resistance to stripping reagents.
Membrane stripping and reprobing (Eaton et al., 2019).
Suitable Detection Systems
Stripping and reprobing work best with chemiluminescent and fluorescent detection methods. In contrast, chromogenic or colorimetric detection reagents leave permanent visible stains on the membrane that cannot be stripped, making these methods unsuitable for reprobing.
Start with Mild Conditions
It is always recommended to begin stripping with mild conditions to minimize the loss of target proteins. Gentle stripping solutions typically involve low pH and mild detergents, which dissociate antibodies while preserving the integrity of the immobilized proteins. In cases where antibodies form strong antigen-antibody complexes, more stringent conditions (e.g., high temperatures, reducing agents) may be required.
Order of Probing for Different Proteins
If multiple proteins are being probed on the same membrane, it is crucial to start by probing for low-abundance proteins. Each round of stripping may result in some loss of immobilized protein, so low-abundance targets should be detected first to minimize the risk of losing detectable levels of these proteins. This ensures the most sensitive proteins are visualized under optimal conditions before any degradation occurs.
Avoid Avidin-Biotin Conjugates
The avidin-biotin interaction is extremely strong, making it difficult to dissociate once formed. As a result, membranes probed with avidin-conjugated antibodies may not be easily stripped, and such detection systems should be avoided when reprobing is anticipated.
Mild Stripping Method
The mild stripping protocol is generally the first approach to consider when stripping and reprobing western blot membranes. It is particularly useful for antibodies that are weakly bound to their antigens or when there is a need to preserve as much antigen as possible for subsequent rounds of probing. This method leverages low pH conditions and mild detergents to dissociate antibody-antigen complexes without causing substantial antigen loss.
Step-by-step procedure for low pH-based stripping
Preparation of Mild Stripping Buffer
The buffer is prepared using common reagents like glycine, SDS (sodium dodecyl sulfate), and Tween 20. The composition of the buffer is critical for ensuring effective yet gentle antibody removal.
Buffer Composition:
- Glycine: 15 g
- SDS: 1 g
- Tween 20: 10 mL
- Deionized water: 800 mL
- pH to 2.2 (adjusted using concentrated HCl)
- Finally, bring the volume to 1000 mL with deionized water.
Glycine acts as the key buffering agent at low pH, while SDS and Tween 20 serve as surfactants to disrupt protein-protein interactions. Low pH weakens the electrostatic and hydrogen bonding interactions between the primary antibody and the target antigen, facilitating the release of antibodies from the membrane without aggressively stripping off the antigen.
Optimal Incubation Time and Temperature
Once the membrane is submerged in the mild stripping buffer, gentle agitation is necessary to ensure complete and uniform exposure to the solution. The typical incubation time ranges from 10 to 20 minutes at room temperature. If the antibody-antigen pair exhibits higher affinity, incubation at 37°C for an additional 5 to 10 minutes may be necessary to achieve effective stripping. It is important to strike a balance between stripping time and temperature to prevent antigen degradation or loss.
Post-stripping Washes
After the stripping incubation, the membrane should be washed thoroughly to remove residual buffer and antibody fragments. Wash the membrane three times for 5 minutes each with TBST (Tris-buffered saline with Tween 20) or PBST (phosphate-buffered saline with Tween 20), with continuous agitation. These washes ensure that the stripping agents and any remaining antibodies are completely removed from the membrane surface.
Reblocking the Membrane
Once the membrane is stripped and washed, it must be reblocked before reprobing. Reblocking serves to prevent non-specific binding during subsequent antibody incubation. The same blocking buffer used in the initial probing (e.g., 5% non-fat milk in TBST or 5% BSA in TBST) should be applied for 1 hour at room temperature. Proper reblocking is essential to reduce background noise in the reprobed blot and ensure clear, specific signals.
This mild method is ideal for situations where the antigen is fragile, low in abundance, or prone to degradation under harsher stripping conditions.
Stringent Stripping Method
For antibodies that form stronger interactions with their target proteins or when mild stripping fails to remove the antibody completely, a more aggressive, stringent stripping protocol is required. This method employs higher temperatures, reducing agents, and more robust detergents to disrupt stronger antigen-antibody bonds.
Step-by-step procedure for stringent stripping using heat and detergent:
Preparation of Stringent Stripping Buffer:
The stringent buffer contains Tris HCl for buffering, SDS for powerful detergent action, and 2-mercaptoethanol as a reducing agent to break disulfide bonds in protein complexes.
Buffer Composition:
- 0.5 M Tris HCl, pH 6.8: 12.5 mL
- 10% SDS: 20 mL
- 2-Mercaptoethanol: 0.8 mL
- Deionized water: 67.5 mL
Due to the volatility and odor of 2-mercaptoethanol, which can be harmful if inhaled, this buffer must be freshly prepared and handled with care. The use of a chemical fume hood is essential for safety during both the preparation and stripping process. SDS, in combination with 2-mercaptoethanol, disrupts hydrophobic and covalent interactions within the antibody-antigen complex, facilitating the removal of even high-affinity antibodies.
Incubation Time and Temperature:
The membrane should be incubated in the stringent buffer at 50°C for 30 minutes with gentle agitation. The elevated temperature aids in the denaturation of proteins and accelerates the reduction of disulfide bonds between the antibody and the antigen. It's important to ensure the membrane is fully submerged in the buffer to guarantee uniform stripping.
Monitoring the incubation time is critical; longer exposure times or higher temperatures can lead to the degradation of the immobilized antigen, particularly for heat-sensitive proteins. However, for highly resilient or strongly bound antibodies, this protocol is often necessary to achieve complete stripping.
Post-stripping Washes:
Following the stripping incubation, the membrane should be washed extensively to remove the stripping buffer and its harsh components. A series of six washes, each lasting 5 minutes, with TBST or PBST is recommended. This thorough washing protocol is crucial for eliminating residual SDS and 2-mercaptoethanol, both of which can interfere with subsequent antibody binding and detection.
Reblocking the Membrane:
After stringent stripping, it is essential to reblock the membrane in the same manner as described in the mild protocol. Due to the harsher conditions of stringent stripping, the membrane's protein-binding sites may become more exposed, making a thorough reblocking step even more important to prevent non-specific antibody binding during reprobe.
Key Considerations for Stringent Stripping:
Antigen Loss Prevention:
While stringent conditions are effective in removing strongly bound antibodies, they also increase the risk of antigen loss or denaturation. Researchers should always start with mild stripping protocols and only escalate to stringent conditions when absolutely necessary. When using stringent stripping, limit the number of reprobing cycles to minimize the cumulative impact on antigen integrity.
Safety Precautions:
The use of 2-mercaptoethanol necessitates strict safety measures, including handling in a well-ventilated fume hood and wearing appropriate personal protective equipment (PPE). This compound emits a strong odor and can be harmful if inhaled, so minimizing exposure is critical.
Troubleshooting Common Issues in Stripping and Reprobing Western Blots
Incomplete Removal of Antibodies
One of the most common issues encountered during stripping is the incomplete removal of the primary or secondary antibodies. This leads to residual signals or ghost bands on the reprobed blot, which can obscure the detection of new targets or complicate quantification.
Symptoms:
- Persistent bands after stripping.
- High background during reprobe, even after blocking.
Causes:
- Inadequate stripping time or insufficient stripping buffer volume.
- Antibodies with strong affinity to the antigen.
- Use of inappropriate stripping conditions (mild conditions when stringent conditions are required).
Solutions:
- Increase stripping time or temperature: Extend the incubation time or increase the temperature slightly (especially for high-affinity antibodies). However, monitor carefully to avoid antigen loss.
- Switch to stringent stripping conditions: If mild stripping is ineffective, use a more stringent protocol that includes higher temperatures and stronger detergents like SDS and reducing agents like 2-mercaptoethanol.
- Perform a control step: After stripping, incubate the membrane with secondary antibody alone to test if primary antibody removal was successful. No signal should be detected if stripping is complete.
Loss of Target Protein (Antigen Degradation or Elution)
Antigen degradation or loss from the membrane can severely limit the ability to reprobe for additional proteins. This is particularly problematic when using sensitive antigens that are susceptible to degradation under harsh conditions.
Symptoms:
- Weak or absent signal upon reprobing.
- Faint or inconsistent bands in the reprobe that were clearly visible in the initial probe.
Causes:
- Overly harsh stripping conditions (high temperature, strong reducing agents).
- Extended stripping time leading to antigen denaturation.
- Use of nitrocellulose membranes instead of more durable PVDF membranes.
Solutions:
- Start with mild stripping conditions: To preserve antigens, always begin with mild stripping protocols and increase stringency only if necessary. Minimize the incubation time and temperature to reduce the risk of antigen loss.
- Use PVDF membranes: PVDF membranes are more robust and have a higher binding capacity for proteins compared to nitrocellulose, making them less prone to protein loss during repeated stripping cycles.
- Limit the number of stripping cycles: The more times a membrane is stripped, the greater the risk of losing antigen. Limit reprobing cycles to two or three rounds to prevent excessive antigen degradation.
High Background or Non-Specific Binding After Reprobe
High background signals can interfere with the clarity of the western blot and make it difficult to distinguish specific bands from non-specific noise.
Symptoms:
- Increased background after reprobing.
- Faint or diffuse bands overlaying the true signal.
- Non-specific binding of secondary antibodies.
Causes:
- Insufficient washing after stripping or blocking.
- Depletion of blocking reagent efficacy.
- Over-saturation of the blot with chemiluminescent substrate or excess antibody concentrations.
Solutions:
- Increase washing steps: After stripping, perform multiple, thorough washes (3-6 times) to remove any residual stripping buffer, antibodies, and detergent. Insufficient washing can leave behind non-specific binders that increase background.
- Reblock the membrane thoroughly: Always reblock the membrane after stripping. Insufficient blocking can lead to non-specific binding, increasing background noise. A fresh blocking solution (e.g., 5% non-fat milk or BSA in TBST) should be applied for at least 1 hour.
- Optimize antibody concentrations: Use optimized, lower concentrations of primary and secondary antibodies to avoid over-saturation of the membrane. Excessive antibody concentrations can increase non-specific binding and background noise.
Inconsistent or Fading Signal After Reprobing
Repeated stripping and reprobing can sometimes lead to weakened signals or variability in band intensity, affecting the ability to quantitatively compare blots.
Symptoms:
- Signals that were strong in the initial probe become faint after reprobing.
- Uneven signal intensities across the blot.
Causes:
- Loss of antigen due to repeated stripping cycles.
- Antibody-antigen interactions may become weaker over multiple rounds of probing.
- Chemiluminescent substrate depletion or inconsistent exposure times.
Solutions:
- Minimize stripping cycles: Limit the number of reprobing rounds to reduce the risk of antigen loss. If you need to detect multiple proteins, consider using different detection channels (e.g., fluorescent secondary antibodies) instead of repeated stripping.
- Use fresh chemiluminescent substrate: If the signal appears faded after reprobe, ensure that fresh chemiluminescent substrate is used to enhance detection. Old or depleted substrate can lead to poor signal intensity.
- Standardize exposure times: When comparing signals across multiple reprobing cycles, maintain consistent exposure times and conditions to ensure accurate comparison of band intensities.
Blot Deterioration or Fragility
Western blot membranes, especially nitrocellulose, can become fragile and prone to tearing after multiple rounds of stripping and washing. This issue is particularly prevalent when handling large or delicate blots.
Symptoms:
- Tears, cracks, or uneven sections on the membrane after stripping.
- Membrane becomes more brittle after multiple reprobing cycles.
Causes:
- Use of nitrocellulose membranes, which are less durable than PVDF.
- Repeated handling and exposure to harsh stripping conditions.
Solutions:
- Switch to PVDF membranes: PVDF membranes are more robust and can withstand multiple rounds of stripping and reprobing without significant degradation. They are also less prone to tearing than nitrocellulose membranes.
- Handle with care: Minimize direct handling of the membrane during washes and incubation steps. Always use forceps or membrane holders to reduce physical stress on the blot.
Residual Chemiluminescent Substrate Interference
In some cases, residual chemiluminescent substrate from the initial detection step can persist on the membrane and interfere with subsequent detection rounds, leading to spurious or unclear results.
Symptoms:
- Persistent faint signals even after stripping.
- Interference with new detection signals, leading to unclear or overlapping bands.
Causes:
- Incomplete removal of chemiluminescent substrate during stripping and washing steps.
- Highly sensitive substrates that leave residual products on the membrane.
Solutions:
- Rinse the membrane thoroughly in water before stripping: Always perform an initial rinse in distilled water to remove excess chemiluminescent substrate before applying the stripping buffer. This helps eliminate background signals that could interfere with reprobing.
- Perform a control reprobe without primary antibody: After stripping, reprobe the membrane with only the secondary antibody. If residual signals persist, this indicates incomplete removal of the substrate or antibodies, and further washing or more stringent stripping may be required.
References
- Eaton, Samantha L., et al. "A guide to modern quantitative fluorescent western blotting with troubleshooting strategies." JoVE (Journal of Visualized Experiments) 93 (2014): e52099.
- Kiyatkin, Anatoly, and Edita Aksamitiene. "Multistrip western blotting to increase quantitative data output." Protein Blotting and Detection: Methods and Protocols (2009): 149-161.
