Enhanced WB Stripping Buffer

Boster’s Enhanced WB Stripping Buffer is a ready-to-use solution for removing primary and secondary antibodies from probed Western blot membranes to allow chemiluminescent Western blots to be reprobed.

Product Info Summary

SKU AR0196
Pack size 500 mL (for 25 assays for an 8× 10cm blot)
Applications Reuse a nitrocellulose or PVDF blot to detect a different target with a different primary antibody
Reprobe a blot to correct or optimize antibody concentrations that were ineffective the first time

Overview

Product Name Enhanced WB Stripping Buffer
SKU/Catalog Number AR0196
Form Liquid
Pack Size 500 mL (for 25 assays for an 8× 10cm blot)
Storage and stability Upon receipt store at 4°C. It is stable for one year.
Note: Precipitant might appear at low temperature. Dissolve the precipitant with water bath before use.
Toxity Nontoxic
Applications Reuse a nitrocellulose or PVDF blot to detect a different target with a different primary antibody
Reprobe a blot to correct or optimize antibody concentrations that were ineffective the first time
Equivalent Thermofisher (Product No. 21059); Millipore Sigma (Product No. S4324)
Description Boster’s Enhanced WB Stripping Buffer is a ready-to-use solution for removing primary and secondary antibodies from probed Western blot membranes to allow chemiluminescent Western blots to be reprobed.
Cite This Product Enhanced WB Stripping Buffer (Boster Biological Technology, Pleasanton CA, USA, Catalog # AR0196)

Assay Principle

Enhanced WB Stripping Buffer allows removing primary and secondary antibodies from probed Western blot membranes without removing or damaging the immobilized antigen. It permits more time efficient experiments that use less sample by reusing membrane without having to re-run gels and blots. Using PVDF membrane will minimize loss of sample protein when stripping antibodies. This product is suitable for the membrane probed with HRP conjugated secondary antibody and detected with chemiluminescent substrate. Protein of lower expression level is recommended to be detected first, applying with Enhanced WB Stripping Buffer, after stripping, protein of higher expression level is then detected.

Features

• Conserve valuable samples - probe the same blot multiple times

• Fast - blot stripped and ready for re-probing in 30 minutes

• Gentle - antibodies removed without harsh reagents or elevated temperatures

• Save time - no need to re-run gels and perform duplicate blots

• Versatile - compatible with both nitrocellulose and PVDF membranes and with a variety of chemiluminescent detection reagents

Additional Materials Required

Chemiluminescent substrate

Tris-buffered saline (TBS) or phosphate-buffered saline (PBS) with 0.05% Tween-20

Primary and secondary antibodies for both first and second Western blotting experiments

Film, Imaging instrument for detecting the chemiluminescent signal

Assay Protocol

Note: Blots may be stored in PBS or TBS at 4°C until the stripping procedure can be performed.

1. Warm the Enhanced WB Stripping Buffer to room temperature.

2. Wash the blot in TBS-T or PBS-T three times for 10 minutes each.

3. Place the blot in Enhanced WB Stripping Buffer and incubate for 20-30 minutes at room temperature. Use a sufficient volume to ensure that the blot is completely wetted (i.e., ~20mL required for a 8× 10cm blot).
Note: Optimization of both incubation time and temperature is essential for best results. For some antibodies, room temperature incubation is sufficient. However, high-affinity antibodies may require incubation for an additional 5-10 minutes at 37°C.

4. Remove the blot from the Enhanced WB Stripping Buffer and wash in TBS-T or PBS-T three times for 10 minutes each.

5. Test for the removal of the immunodetection reagents as follows:
• Test for complete removal of the secondary antibody: Incubate the membrane with ECL Western Blotting Substrate and expose to film. If no signal is detected, the secondary antibody has been successfully removed from the antigen or primary antibody.
• Test for complete removal of the primary antibody: Incubate the membrane with the HRP-labeled secondary antibody, followed by a wash in wash buffer. Incubate the membrane with ECL Western Blotting Substrate and expose to film. If no signal is detected, the primary antibody has been successfully removed from the antigen.

6. If signal is detected with either test in step 5, return to step 2, stripping for an additional 5-15 minutes. Some antigen/antibody systems require increased temperature and/or longer incubation times to strip them fully. Optimize stripping time and temperature to ensure complete removal of antibodies while preventing damage to the antigen.

7. After determining that the membrane is properly stripped, the second immunoprobing experiment may be performed.

Notes:
• Blot can be stripped and reprobed several times but might require longer exposure times or a more sensitive chemiluminescent substrate. Subsequent reprobings might result in decreased signal if the antigen is labile in Enhanced Western Blot Stripping Buffer. Analysis of the individual system is required.
• Reblocking the membrane is recommended after stripping.

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