Download Technical Tip: Stripping and reprobing western blots
Learn about our specially formulated buffers for every step of western blot processing and detection. We offer a wide range of blocking buffers, wash buffers, detergents, and western blot stripping buffers to help generate the best data possible from your western blots.
Recommended western blot blocking buffers to optimize the detection of your target proteins
| Blocking buffer | Blocking agent | Highlights | When to use | Available formats |
|---|---|---|---|---|
| Chemiluminescent western blotting | ||||
| StartingBlock Blocking Buffer | Serum- and biotin-free single purified protein |
|
| PBS TBS PBST TBST |
| Blocker Casein | Purified protein |
|
| PBS TBS |
| Blocker BSA | Purified bovine serum albumin |
|
| PBS TBS |
| Fluorescent western blotting | ||||
| Blocker FL | Single purified protein |
|
| 10X concentrate |
View all available western blot blocking buffers.
Before probing for proteins of interest, the remaining binding surface of the membrane must be blocked to prevent the nonspecific binding of the antibodies. Otherwise, the antibodies or other detection reagents will bind to any remaining sites on the membrane that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking.
A variety of blocking buffers ranging from milk or normal serum to highly purified proteins can be used to block free sites on a membrane. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding. Blocking buffers can influence antibody binding and specificity- so optimization is needed. No single protein or mixture of proteins works best for all western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system.
Choose from dry blend packs or pouches of pre-blended powder mixtures of commonly-needed buffers, such as PBS and TBS for western blot methods.
| TBS | PBS | TBST | PBST | |
|---|---|---|---|---|
| Dry blend | BupH Tris Buffered Saline Packs (Cat. No. 28376 and 28379 - 40, 10 pks) | BupH Phosphate Buffered Saline Packs (Cat. No. 28372 - 40 pks) | ||
| Liquid conc. | Pierce 20X TBS Buffer (Cat. No. 28358 - 500ml) | Pierce 20X Phosphate Buffered Saline (Cat. No. 28348 - 500ml) | Pierce 20X TBS Tween 20 Buffer (Cat. No. 28360 - 500ml) | Pierce 20X PBS Tween 20 Buffer (Cat. No. 28352 - 500ml) |
| Formulation |
|
|
|
|
Washing steps are necessary through the series of incubations to remove unbound reagents and reduce background. Insufficient washing produces high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot.
A variety of buffers may be used. Washing is performed in physiological buffers such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS). Detergents such as Tween-20 can be added to the buffer to help remove nonspecifically bound material. The amount of Tween-20 (0.05%-0.2%) will vary depending on the strength of the antibodies used. Weak binding antibodies may be washed away by too much detergent.
In applications where alkaline phosphatase conjugates are used, TBS should be selected as PBS interferes with alkaline phosphatase. When performing fluorescent western blotting, it is recommended to eliminate the detergent from the blocking step. Detergents auto-fluoresce and will contribute to higher background.
Choosing the right buffer for your need
| Tween-20 | TBS | PBS | |
|---|---|---|---|
| HRP conjugated systems | Y | Y | Y |
| AP conjugated systems | Y | Y | N |
| Fluorescent systems | N (can be used after blocking step) | Y | Y |
Western blot stripping buffers
Reprobing a western blot saves time and conserves sample while allowing optimization to be performed as needed. These specially formulated stripping buffers are designed to dissociate and strip primary and secondary antibodies from western blots so that membranes can be reprobed under alternate conditions or with another antibody to detect a different protein target.
| Restore Stripping Buffer | Restore Plus Stripping | Restore Fluorescent Western Blot Stripping Buffer | |
|---|---|---|---|
| Features |
|
|
|
| Membrane | NC and PVDF | NC and PVDF | Use with low fluorescence PVDF membranes (e.g., 22860) |
| Time of incubation | 15-30 min at 37°C | 5-15 mins at RT or 37°C for high affinity antibodies | 10-20 min at RT |
| When to use | If primary antibody is susceptible to stripping buffers | To remove high-affinity primary antibodies | To remove NIR-labeled antibodies |
| Cat. No. | 21059 (500 mL) 21063 (5 L) | 46430 (500 mL) | 62300 (100 mL) |
Advantages of stripping and reprobing blots
There are several major reasons to choose to strip and reprobe a western blot. Following are some of the most important:
| Conserves sample | When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed with the same or different antibodies. |
| Saves time | It is time-consuming to run an SDS-polyacrylamide gel and then transfer the proteins to a membrane. By using the same blot for several different detections, you save time. |
| Saves money | By reusing the same blot, you save money on the costs of membrane, buffers and protein sample. |
| Assay optimization is easier | The light emission intensity of high sensitivity chemiluminescent substrates often require antibody concentration optimization to achieve the highest quality blot. Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration. |
| Quickly confirm atypical results | When immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back to gel electrophoresis. |
| Correct mistakes | Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. By stripping the membrane, the blot can be reused. |
Signal enhancements
| SuperSignal Western Blot Enhancer | |
|---|---|
 | Need: Protein is difficult to detect because of low abundance or poor immunoreactivity Solution: Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to a detection performed without it. |
| Catalog No. | 46640 |
| Clean-Blot IP Detection Reagent | |
|---|---|
 | Need: Obstructed detection of target protein due to interference from both heavy-chain and light-chain IgG fragments from immunoprecipitation assay Solution: HRP conjugate that is optimized for post-immunoprecipitation western blot detection of primary antibodies without interference from denatured immunoprecipitation (IP) antibody fragments. |
| Catalog No. | 21230 (HRP Reagent only) 21232 (Clean-Blot IP Detection Kit) |
Blocking buffers
Wash buffers and detergents
Stripping buffers
Blocking buffers
Stripping buffers
Application notes and technical handbooks
Recommended western blot blocking buffers to optimize the detection of your target proteins
| Blocking buffer | Blocking agent | Highlights | When to use | Available formats |
|---|---|---|---|---|
| Chemiluminescent western blotting | ||||
| StartingBlock Blocking Buffer | Serum- and biotin-free single purified protein |
|
| PBS TBS PBST TBST |
| Blocker Casein | Purified protein |
|
| PBS TBS |
| Blocker BSA | Purified bovine serum albumin |
|
| PBS TBS |
| Fluorescent western blotting | ||||
| Blocker FL | Single purified protein |
|
| 10X concentrate |
View all available western blot blocking buffers.
Before probing for proteins of interest, the remaining binding surface of the membrane must be blocked to prevent the nonspecific binding of the antibodies. Otherwise, the antibodies or other detection reagents will bind to any remaining sites on the membrane that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target or probe components in the assay can be used for blocking.
A variety of blocking buffers ranging from milk or normal serum to highly purified proteins can be used to block free sites on a membrane. The ideal blocking buffer will bind to all potential sites of nonspecific interaction, eliminating background altogether without altering or obscuring the epitope for antibody binding. Blocking buffers can influence antibody binding and specificity- so optimization is needed. No single protein or mixture of proteins works best for all western blot experiments, and empirical testing is necessary to obtain the best possible results for a given combination of specific antibodies, membrane type and substrate system.
Choose from dry blend packs or pouches of pre-blended powder mixtures of commonly-needed buffers, such as PBS and TBS for western blot methods.
| TBS | PBS | TBST | PBST | |
|---|---|---|---|---|
| Dry blend | BupH Tris Buffered Saline Packs (Cat. No. 28376 and 28379 - 40, 10 pks) | BupH Phosphate Buffered Saline Packs (Cat. No. 28372 - 40 pks) | ||
| Liquid conc. | Pierce 20X TBS Buffer (Cat. No. 28358 - 500ml) | Pierce 20X Phosphate Buffered Saline (Cat. No. 28348 - 500ml) | Pierce 20X TBS Tween 20 Buffer (Cat. No. 28360 - 500ml) | Pierce 20X PBS Tween 20 Buffer (Cat. No. 28352 - 500ml) |
| Formulation |
|
|
|
|
Washing steps are necessary through the series of incubations to remove unbound reagents and reduce background. Insufficient washing produces high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot.
A variety of buffers may be used. Washing is performed in physiological buffers such as Tris-buffered saline (TBS) or phosphate-buffered saline (PBS). Detergents such as Tween-20 can be added to the buffer to help remove nonspecifically bound material. The amount of Tween-20 (0.05%-0.2%) will vary depending on the strength of the antibodies used. Weak binding antibodies may be washed away by too much detergent.
In applications where alkaline phosphatase conjugates are used, TBS should be selected as PBS interferes with alkaline phosphatase. When performing fluorescent western blotting, it is recommended to eliminate the detergent from the blocking step. Detergents auto-fluoresce and will contribute to higher background.
Choosing the right buffer for your need
| Tween-20 | TBS | PBS | |
|---|---|---|---|
| HRP conjugated systems | Y | Y | Y |
| AP conjugated systems | Y | Y | N |
| Fluorescent systems | N (can be used after blocking step) | Y | Y |
Western blot stripping buffers
Reprobing a western blot saves time and conserves sample while allowing optimization to be performed as needed. These specially formulated stripping buffers are designed to dissociate and strip primary and secondary antibodies from western blots so that membranes can be reprobed under alternate conditions or with another antibody to detect a different protein target.
| Restore Stripping Buffer | Restore Plus Stripping | Restore Fluorescent Western Blot Stripping Buffer | |
|---|---|---|---|
| Features |
|
|
|
| Membrane | NC and PVDF | NC and PVDF | Use with low fluorescence PVDF membranes (e.g., 22860) |
| Time of incubation | 15-30 min at 37°C | 5-15 mins at RT or 37°C for high affinity antibodies | 10-20 min at RT |
| When to use | If primary antibody is susceptible to stripping buffers | To remove high-affinity primary antibodies | To remove NIR-labeled antibodies |
| Cat. No. | 21059 (500 mL) 21063 (5 L) | 46430 (500 mL) | 62300 (100 mL) |
Advantages of stripping and reprobing blots
There are several major reasons to choose to strip and reprobe a western blot. Following are some of the most important:
| Conserves sample | When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed with the same or different antibodies. |
| Saves time | It is time-consuming to run an SDS-polyacrylamide gel and then transfer the proteins to a membrane. By using the same blot for several different detections, you save time. |
| Saves money | By reusing the same blot, you save money on the costs of membrane, buffers and protein sample. |
| Assay optimization is easier | The light emission intensity of high sensitivity chemiluminescent substrates often require antibody concentration optimization to achieve the highest quality blot. Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration. |
| Quickly confirm atypical results | When immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back to gel electrophoresis. |
| Correct mistakes | Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. By stripping the membrane, the blot can be reused. |
Signal enhancements
| SuperSignal Western Blot Enhancer | |
|---|---|
| Need: Protein is difficult to detect because of low abundance or poor immunoreactivity Solution: Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to a detection performed without it. |
| Catalog No. | 46640 |
| Clean-Blot IP Detection Reagent | |
|---|---|
| Need: Obstructed detection of target protein due to interference from both heavy-chain and light-chain IgG fragments from immunoprecipitation assay Solution: HRP conjugate that is optimized for post-immunoprecipitation western blot detection of primary antibodies without interference from denatured immunoprecipitation (IP) antibody fragments. |
| Catalog No. | 21230 (HRP Reagent only) 21232 (Clean-Blot IP Detection Kit) |
Blocking buffers
Wash buffers and detergents
Stripping buffers
Blocking buffers
Stripping buffers
Application notes and technical handbooks
How-to videos
How to probe a western blot
See the steps involved in western blot analysis after transferring your proteins to a membrane.
How to strip and reprobe your western blots
Learn how to reprobe your western blot using Restore Stripping Buffers.
Resources
For Research Use Only. Not for use in diagnostic procedures.