Regarding the issue you encountered with ineffective antibody stripping in Western Blot, based on our experimental experience (MedChemExpress,MCE) and literature references, we recommend the following optimized protocol:

Recommended Buffer Formulation: Through systematic comparison, we found that a strongly denaturing buffer system containing SDS and β-mercaptoethanol (e.g., 62.5 mM Tris-HCl pH 6.8, 2% SDS, 100 mM β-mercaptoethanol) significantly improves antibody stripping efficiency when incubated at 65°C. This formulation is based on an optimized protocol published in Analytical Biochemistry (PMID: 19303392), where high concentrations of denaturing agents effectively dissociate antigen-antibody complexes.

Key Operational Parameters:

• Temperature control: Incubation in a 65°C water bath for 30–40 minutes is recommended, as elevated temperature enhances protein denaturation.
• Utilization of molecular weight differences: This protocol is particularly effective when the target protein and interfering proteins differ by ≥10 kDa.

Notes:

• This buffer produces a noticeable thiol odor, so it is advisable to handle it in a fume hood.
• For membranes reused multiple times, residual SDS may affect subsequent experiments; therefore, increasing TBST washing steps (5 × 5 minutes) is recommended.

This protocol enhances both protein denaturation and disulfide bond reduction, addressing the limitations of conventional stripping buffers in completely removing antibodies. It is especially suitable for precious samples requiring repeated detection.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

[email protected]

https://www.abcam.com/en-us/technical-resources/guides/western-blot-guide/membrane-stripping-for-western-blot?srsltid=AfmBOoru4o08-eWM1yE0a9ADbJlnrlPWmQ--tkbAqUdIxAed9nK6cYxZ

this mild stripping protocol has never failed for me.