Testing Data
Enhanced WB Reagent
Enhanced WB Stripping Buffer
Synonyms
Enhanced WB; Enhanced WB Stripping Buffer; Enhanced WB reagent
Form/Format
Liquid
Application Notes
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.
Reprobe a blot to correct or optimize antibody concentrations that were ineffective the first time.
Toxity
Nontoxic
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
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
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 degree 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 degree 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.
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 degree 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.
Preparation and Storage
Upon receipt store at 4 degree C. It is stable for one year.
Note: Precipitant might appear at low temperature. Dissolve the precipitant with water bath before use.
Note: Precipitant might appear at low temperature. Dissolve the precipitant with water bath before use.
Testing Data
Testing Data
Related Product Information for Enhanced WB reagent
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 Categories/Family for Enhanced WB reagent
Similar Products
Product Notes
The Enhanced WB (Catalog #AAA1753879) is a Reagent and is intended for research purposes only. The product is available for immediate purchase. 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. Researchers should empirically determine the suitability of the Enhanced WB for an application not listed in the data sheet. Researchers commonly develop new applications and it is an integral, important part of the investigative research process. It is sometimes possible for the material contained within the vial of "Enhanced WB, Reagent" to become dispersed throughout the inside of the vial, particularly around the seal of said vial, during shipment and storage. We always suggest centrifuging these vials to consolidate all of the liquid away from the lid and to the bottom of the vial prior to opening. Please be advised that certain products may require dry ice for shipping and that, if this is the case, an additional dry ice fee may also be required.Precautions
All products in the AAA Biotech catalog are strictly for research-use only, and are absolutely not suitable for use in any sort of medical, therapeutic, prophylactic, in-vivo, or diagnostic capacity. By purchasing a product from AAA Biotech, you are explicitly certifying that said products will be properly tested and used in line with industry standard. AAA Biotech and its authorized distribution partners reserve the right to refuse to fulfill any order if we have any indication that a purchaser may be intending to use a product outside of our accepted criteria.Disclaimer
Though we do strive to guarantee the information represented in this datasheet, AAA Biotech cannot be held responsible for any oversights or imprecisions. AAA Biotech reserves the right to adjust any aspect of this datasheet at any time and without notice. It is the responsibility of the customer to inform AAA Biotech of any product performance issues observed or experienced within 30 days of receipt of said product. To see additional details on this or any of our other policies, please see our Terms & Conditions page.Item has been added to Shopping Cart
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