Western blot stripping removes primary and secondary antibodies from a previously probed membrane. This prepares the membrane for re-probing, allowing analysis of additional proteins on the same sample. It allows sequential detection of other target proteins, preventing interference from previous signals.
Purpose of Stripping
Western blot stripping serves several purposes in molecular biology. It conserves valuable samples by allowing multiple analyses from a single blot, reducing the need for new gels and transfers. This also reduces experimental variability by using the same protein transfer for different targets. Researchers often strip blots to confirm initial results, re-analyze anomalous findings, or probe for loading control proteins, which are used to ensure consistent protein loading across samples. It also allows for the optimization of antibody concentrations, saving time and reagents.
Methods and Protocols for Stripping
Different methods exist for stripping Western blot membranes, categorized by chemical approach or harshness. Low pH buffers, such as glycine-HCl, are used as a milder method. They alter antibody structure, causing dissociation from target proteins. A typical low pH stripping solution might include 25-200 mM glycine, 0.1-1% SDS, and optionally 1% Tween 20, adjusted to a pH of 2.0-2.2.
For robust antibody removal, especially those with strong binding, high pH buffers or those combining heat and detergents are used. These “harsh” methods often involve sodium dodecyl sulfate (SDS) and reducing agents such as beta-mercaptoethanol (βME) or dithiothreitol (DTT), which denature antibodies and disrupt antigen interaction. A common harsh stripping buffer recipe includes 2% SDS, 62.5 mM Tris-HCl (pH 6.8), and 100 mM (0.8%) βME, often incubated around 50°C. Commercial stripping solutions are also available, offer convenience and faster stripping times, typically within 15 to 30 minutes, without requiring high temperatures or strong reducing agents.
A general stripping protocol involves several steps. After initial detection, wash the membrane with a buffer (e.g., Tris-buffered saline with Tween 20 (TBST) or phosphate-buffered saline (PBS)) to remove residual reagents. Incubate the membrane in the chosen stripping buffer, with gentle agitation, for a period ranging from 10 minutes to over an hour, depending on the buffer and antibody affinity. After incubation, wash extensively to remove stripping buffer components, especially reducing agents like βME, which can interfere with subsequent antibody binding. Re-block the membrane to prevent non-specific binding before re-probing with new primary and secondary antibodies.
Optimizing and Troubleshooting Stripping
Successful Western blot stripping depends on several factors. The strength of antibody-antigen binding impacts antibody removal; stronger interactions require harsher stripping conditions or longer incubation. Membrane type also plays a role. Polyvinylidene difluoride (PVDF) membranes retain proteins better and are more suitable for multiple stripping cycles than nitrocellulose, which can experience greater protein loss. Duration and temperature are important; longer times or higher temperatures can improve antibody removal but may also lead to increased protein loss from the membrane.
Optimizing the stripping process balances effective antibody removal with protein preservation. Researchers typically begin with milder stripping conditions and gradually increase stringency if residual signal persists. It is beneficial to test different stripping conditions, such as varying incubation times or temperatures, to find the optimal balance for specific antibodies and proteins. Ensuring thorough washing steps after stripping is paramount to remove all traces of stripping buffer, which could otherwise denature new antibodies or cause high background signals during re-probing. When planning to detect multiple proteins, it is often advisable to probe for low-abundance proteins first, as some protein loss can occur with each stripping cycle, potentially making low-abundance targets undetectable later.
Common issues encountered during stripping include incomplete antibody removal, which can result in “ghost bands” from the previous detection, or a significant loss of target protein signal after re-probing. If incomplete stripping occurs, solutions involve increasing the concentration of detergents or reducing agents in the buffer, elevating the incubation temperature (e.g., to 50°C), or extending the stripping time. Conversely, if protein signal is lost, the stripping conditions might be too harsh; switching to a milder buffer, reducing incubation time, or lowering the temperature can help preserve protein integrity. High background signal after stripping can often be resolved by optimizing the blocking conditions or performing more rigorous washes between steps. Using fresh reagents, especially volatile ones like beta-mercaptoethanol, is also important for consistent and effective stripping.