Western blot buffers

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 membrane stripping buffers to help generate the best data possible from your western blots.

Explore our variety of western blot blocking buffers to optimize the detection of your target proteins

Blocking buffer Blocking agent Highlights When to use Available formats
StartingBlock
  • Serum and biotin-free single purified protein
  • Performs well with a wide range of antibodies and antibody combinations.
  • Compatible with streptavidin systems
  • Blocks in less than 15 minutes
  • Best for med-high abundant proteins or strong antibody affinity
  • High background with current blocking buffer
  • Stripping and reprobing western blots
PBS
TBS
PBST
TBST
Blocker FL
  • Single purified protein
  • Blocks excess nonspecific binding sites to help reduce background fluorescence,
  • Works with both nitrocellulose and low fluorescence PVDF
  • Detergent-free
  • Blocks in 15-30 minutes
  • Fluorescence western blotting
  • Imaging and storage of dry fluorescence blots
10X
Pierce Clear Milk Blocking Buffer
  • Clarified and stabilized milk proteins
  • High performance replacement for homemade milk blocking buffers in Western blotting applications
  • Long shelf-life at room temperature
  • Use when high background seen with Non-fat Milk
  • Fluorescent and chemiluminescent applications
Borate, pH 7.6
SuperBlock
  • Serum and biotin-free single purified glycoprotein
  • Protein-based formulation does not contain any immunoglobulins, albumin or endogenous biotin, making it compatible in many situations where traditional blocking agents fail.
  • Biotin-free for use with streptavidin system
  • Blocks in less than 10 minutes
  • High background with current blocking buffer
PBS
TBS
PBST
TBST
SEA BLOCK Blocking Buffer
  • Steelhead salmon serum
  • Useful in detection methods involving mammalian samples.
  • Particularly effective in applications involving fluorescence imaging
  • Mammalian samples
  • Fluorescence western blotting
PBS
Blocker BSA
  • Purified bovine serum albumin
  • 10% solutions of high-quality bovine serum albumin
  • Single purified protein provides fewer chances of cross-reaction with assay components than serum or milk solutions
  • Use when targeting phospho- proteins
  • Best to use when storing reused antibodies in blocker
PBS
TBS
Blocker Caesin
  • Purified casein
  • Single-protein blocking buffer provides fewer chances of cross-reaction with assay components than serum or milk solutions
  • Use when high background seen with Non-fat milk blockers
PBS
TBS
Blocker BLOTTO
  • Non-fat dry milk
  • Ready-to-use 5% solution of nonfat powdered milk
  • Convenience- ready-to-use
  • More consistent product over home-made blockers
TBS
Protein-Free
  • Non-protein blocking compound
  • Minimizes or eliminates cross-reactivity associated with protein-based blocking buffers.
  • Sample-and-antibody combinations require the elimination of all possible exogenous animal proteins in the assay system to avoid cross-reaction or quenching of the desired probe function
  • Use when protein-based blockers cause high background
PBS
TBS
PBST
TBST
Pierce Fast Blocking Buffer
  • Single purified protein
  • Blocks in 5 minutes
  • When time is essential
TBS
SuperSignal Western Blot Enhancer
  • Membrane treatment for low abundance or poor immunoreactivity antibodies
  • Pre-treatment for nitrocellulose membranes
  • Reduces the amount of primary antibody required for probing
  • Use when primary antibody is 3 to 100-fold less primary antibody than is usually used to detect the protein of interest
Ready-to-use

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
  • 25mM Tris
  • 0.15M NaCl
  • pH 7.2
  • 10mM sodium phosphate
  • 0.15M NaCl
  • pH 7.5
  • 25mM Tris
  • 0.15M NaCl
  • 0.05% Tween-20
  • pH 7.5
  • 10mM sodium phosphate
  • 0.15M NaCl
  • 0.05% Tween-20
  • pH 7.5

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

Reprobing a western blot saves time and conserves sample while allowing optimization to be performed as needed. These specially formulated 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
  • Gentle, odor-free
  • Robust yet gentle, odor free
  • Designed for use with antibodies that are difficult to remove from Western blots, require longer incubation times, or incubation temperatures greater than 22°C
Gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from Western blots
Membrane NC and PVDF NC and PVDF Use with low fluorescence PVDF membranes (e.g. 22860)
Time of incubation 15-30 min at RT 5-15 mins at RT 15 min at RT
Select when… Primary antibody is susceptible to stripping buffers Removing high affinity primary antibodies Removing NIR labeled antibodies

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.

Blocking buffers

Wash buffers and detergents

Stripping buffers

Explore our variety of western blot blocking buffers to optimize the detection of your target proteins

Blocking buffer Blocking agent Highlights When to use Available formats
StartingBlock
  • Serum and biotin-free single purified protein
  • Performs well with a wide range of antibodies and antibody combinations.
  • Compatible with streptavidin systems
  • Blocks in less than 15 minutes
  • Best for med-high abundant proteins or strong antibody affinity
  • High background with current blocking buffer
  • Stripping and reprobing western blots
PBS
TBS
PBST
TBST
Blocker FL
  • Single purified protein
  • Blocks excess nonspecific binding sites to help reduce background fluorescence,
  • Works with both nitrocellulose and low fluorescence PVDF
  • Detergent-free
  • Blocks in 15-30 minutes
  • Fluorescence western blotting
  • Imaging and storage of dry fluorescence blots
10X
Pierce Clear Milk Blocking Buffer
  • Clarified and stabilized milk proteins
  • High performance replacement for homemade milk blocking buffers in Western blotting applications
  • Long shelf-life at room temperature
  • Use when high background seen with Non-fat Milk
  • Fluorescent and chemiluminescent applications
Borate, pH 7.6
SuperBlock
  • Serum and biotin-free single purified glycoprotein
  • Protein-based formulation does not contain any immunoglobulins, albumin or endogenous biotin, making it compatible in many situations where traditional blocking agents fail.
  • Biotin-free for use with streptavidin system
  • Blocks in less than 10 minutes
  • High background with current blocking buffer
PBS
TBS
PBST
TBST
SEA BLOCK Blocking Buffer
  • Steelhead salmon serum
  • Useful in detection methods involving mammalian samples.
  • Particularly effective in applications involving fluorescence imaging
  • Mammalian samples
  • Fluorescence western blotting
PBS
Blocker BSA
  • Purified bovine serum albumin
  • 10% solutions of high-quality bovine serum albumin
  • Single purified protein provides fewer chances of cross-reaction with assay components than serum or milk solutions
  • Use when targeting phospho- proteins
  • Best to use when storing reused antibodies in blocker
PBS
TBS
Blocker Caesin
  • Purified casein
  • Single-protein blocking buffer provides fewer chances of cross-reaction with assay components than serum or milk solutions
  • Use when high background seen with Non-fat milk blockers
PBS
TBS
Blocker BLOTTO
  • Non-fat dry milk
  • Ready-to-use 5% solution of nonfat powdered milk
  • Convenience- ready-to-use
  • More consistent product over home-made blockers
TBS
Protein-Free
  • Non-protein blocking compound
  • Minimizes or eliminates cross-reactivity associated with protein-based blocking buffers.
  • Sample-and-antibody combinations require the elimination of all possible exogenous animal proteins in the assay system to avoid cross-reaction or quenching of the desired probe function
  • Use when protein-based blockers cause high background
PBS
TBS
PBST
TBST
Pierce Fast Blocking Buffer
  • Single purified protein
  • Blocks in 5 minutes
  • When time is essential
TBS
SuperSignal Western Blot Enhancer
  • Membrane treatment for low abundance or poor immunoreactivity antibodies
  • Pre-treatment for nitrocellulose membranes
  • Reduces the amount of primary antibody required for probing
  • Use when primary antibody is 3 to 100-fold less primary antibody than is usually used to detect the protein of interest
Ready-to-use

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
  • 25mM Tris
  • 0.15M NaCl
  • pH 7.2
  • 10mM sodium phosphate
  • 0.15M NaCl
  • pH 7.5
  • 25mM Tris
  • 0.15M NaCl
  • 0.05% Tween-20
  • pH 7.5
  • 10mM sodium phosphate
  • 0.15M NaCl
  • 0.05% Tween-20
  • pH 7.5

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

Reprobing a western blot saves time and conserves sample while allowing optimization to be performed as needed. These specially formulated 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
  • Gentle, odor-free
  • Robust yet gentle, odor free
  • Designed for use with antibodies that are difficult to remove from Western blots, require longer incubation times, or incubation temperatures greater than 22°C
Gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from Western blots
Membrane NC and PVDF NC and PVDF Use with low fluorescence PVDF membranes (e.g. 22860)
Time of incubation 15-30 min at RT 5-15 mins at RT 15 min at RT
Select when… Primary antibody is susceptible to stripping buffers Removing high affinity primary antibodies Removing NIR labeled antibodies

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.

Blocking buffers

Wash buffers and detergents

Stripping buffers

Resources

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