Western blotting is a widely used laboratory method for identifying specific proteins within a sample. It involves separating proteins by size, transferring them to a membrane, and using antibodies for detection. Researchers often need to analyze multiple proteins from the same sample or optimize detection conditions. Membrane stripping allows removal of previously bound antibodies, enabling re-probing of the same membrane. This conserves precious protein samples and saves considerable time.
Purpose of Stripping
Stripping a Western blot membrane offers several practical advantages. It enables re-use of a single membrane for detecting multiple proteins, especially beneficial when sample material is scarce or limited.
Stripping also allows direct comparison of different proteins or modifications on the identical sample lane. This minimizes variability from running multiple gels, ensuring a direct comparison. For instance, it’s commonly used to probe for a protein of interest and then re-probe for a loading control, confirming consistent protein loading. Stripping also assists in optimizing primary antibody concentrations by allowing re-probing with different dilutions if initial detection was not ideal, thereby improving signal quality.
Essential Materials for Stripping
Successfully stripping a Western blot requires specific reagents and equipment. Common stripping buffers often contain components like sodium dodecyl sulfate (SDS), Tris-HCl, and a reducing agent such as beta-mercaptoethanol. SDS helps denature proteins and disrupt antibody-antigen interactions, while reducing agents break disulfide bonds. Some protocols may also use low pH solutions (typically around 2.0-2.8) with glycine to aid in antibody dissociation.
Essential laboratory equipment includes an orbital shaker for gentle agitation, and appropriate containers to ensure the membrane is fully submerged. A water bath may be needed for elevated temperatures. Using a polyvinylidene difluoride (PVDF) membrane is recommended over nitrocellulose, as PVDF is more robust and retains proteins better during stripping.
Step-by-Step Stripping Protocol
The process of stripping and reprobing a Western blot involves several careful steps to ensure effective antibody removal without significant protein loss. After initial detection and imaging, thoroughly wash the membrane with a wash buffer (e.g., TBST) to remove residual detection reagents.
Next, prepare the stripping buffer according to your chosen protocol; mild buffers often contain glycine and SDS at low pH, while harsher options may include SDS and beta-mercaptoethanol with heat. Ensure sufficient buffer volume to fully submerge the membrane in an appropriate container. Incubate the membrane in the stripping buffer with gentle agitation on an orbital shaker. Incubation times range from 10 to 45 minutes, and temperatures can vary from room temperature to 50-70°C, depending on the buffer’s harshness and antibody binding strength.
Following stripping incubation, perform thorough washing steps. Wash the membrane multiple times (typically 3 to 6 times for 5-10 minutes each) with wash buffer to completely remove all traces of stripping buffer components and dissociated antibodies. Incomplete removal of stripping reagents can interfere with subsequent antibody binding. After washing, re-block the membrane with a fresh blocking solution (e.g., milk or BSA) for at least one hour to prevent non-specific antibody binding during the next probing round. The membrane is then ready for incubation with new primary and secondary antibodies.
Common Issues and Solutions
Several issues can arise during stripping and reprobing, potentially affecting subsequent protein detection quality. Incomplete stripping occurs when residual signal from previous antibodies remains visible. This can happen if antibody-antigen interactions are strong or initial stripping conditions were too mild. To address this, increase incubation time or temperature, or switch to a harsher stripping buffer. Confirm successful stripping by re-blocking and incubating with only secondary antibody; no signal indicates effective stripping.
Loss of target protein from the membrane can happen if the stripping process is too harsh, resulting in a weak or absent signal upon reprobing. To mitigate protein loss, reduce stripping time, lower temperature, or use a milder stripping reagent. PVDF membranes are generally more resistant to protein loss during stripping compared to nitrocellulose.
Membrane damage can also occur due to improper handling or excessively harsh stripping conditions, leading to physical tears or uneven areas. Gentle handling with forceps and ensuring full submersion helps prevent damage.
High background noise after reprobing can obscure target protein bands. This can be caused by insufficient washing, inadequate re-blocking, or residual stripping buffer. Solutions include performing more rigorous washes after stripping, optimizing the blocking step, and ensuring all stripping buffer is thoroughly rinsed away before re-blocking and reprobing.