Western Blot Buffers

Thermo scientific western blot buffers including blocking buffers, stripping buffers and wash 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 western blot stripping buffers to help generate the best data possible from your western blots.

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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

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	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 37°C	5-15 mins at RT or 37°C for high affinity antibodies	10-20 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

Blocking buffers

Wash buffers and detergents

Stripping buffers