Before using antibodies to detect proteins that have been dotted or transferred to a membrane, the remaining binding surface must be blocked to prevent the non-specific binding of the antibodies. Otherwise, the antibodies or other detection reagents will bind to any remaining sites that initially served to immobilize the proteins of interest. In principle, any protein that does not have binding affinity for the target protein or probe components (antibodies) in the assay can be used for blocking. In practice, however, certain proteins perform better than others because they bind to the membrane or other immobilization surface more consistently or because they stabilize the function of other system components.

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Purpose and function of blocking steps

The membrane supports, such as nitrocellulose and polyvinylidene diflurode (PVDF), used in western blotting have a high affinity for proteins. To prevent non-specific binding of detection antibodies during the steps following transfer, unoccupied sites on the membrane surface must be blocked. Blocking buffer formulations vary widely and may contain milk, normal serum, or highly purified proteins to block free membrane sites. The blocking step is imperative and improves the signal-to-noise ratio of the assay by reducing background interference. Inadequate amounts of blocker result in excessive background noise and a reduced signal-to-noise ratio; in contrast, excessive concentrations of blocker may mask antibody-antigen interactions or inhibit the marker enzyme, which causes a reduction of the target signal.

Types of blocking buffers

Several types of blocking buffers have been successfully used in western blotting. A majority of western blot blocking buffers are inert solutions of either mixed proteins or a single purified protein that ideally have little to no interaction with the detection antibodies or antigens on the blot. Typically, blocking agents are diluted in either Tris-buffered saline (TBS) or phosphate-buffered saline (PBS), with or without detergent. Detergents, such as Tween-20, can be added to the blocking buffer to further reduce non-specific binding. 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 subsequent washes.

Blocking Buffer/AgentBenefitsConsiderations
2-5% Non-fat milkInexpensive, contains multiple types of proteinsContains biotin and phosphoproteins, which can interfere with streptavidin-biotin detection strategies and detection of phosphorylated target proteins. Due to the number of proteins within milk, milk may mask some antigens and lower the detection limit of the western blot.
2-3% Bovine serum albumin (BSA)Good alternative to milk, can be used in biotin-streptavidin systems or when probing for phosphoproteinsVarious grades of BSA are commercially available that can impact signal-to-noise. BSA is generally a weaker blocker, which can result in more non-specific antibody binding, but can increase the detection sensitivity for low-abundant proteins.
Purified proteins (e.g., casein)Single-protein blocking buffers can provide fewer chances of cross-reaction with assay components than serum or milk solutions. Ideal when blockers, such as non-fat milk, block antigen-antibody bindingMore expensive than traditional non-fat milk formulations.

Which blocking buffer to use?

Selection of blocking buffer for western blotting applications is often system-dependent. Determining the proper blocking buffer can help to increase the system’s signal-to-noise ratio. Occasionally, when switching from one substrate to another, the blocking buffer may need to be changed in order to avoid problems with diminished signal or increased background. For example, with applications using an alkaline phosphatase (AP) conjugate, a blocking buffer in Tris-buffered saline (TBS) should be selected because phosphate-buffered saline (PBS) interferes with AP activity. Empirically testing various blocking buffers for use with a given system can help achieve the best possible results. No single blocking agent is ideal for every application because each antibody-antigen pair has unique characteristics.

The accompanying figures illustrate the value of testing different blocking buffers as part of western blotting optimization. In these example experiments, in which all other conditions were equal, different blocking buffers quenched or enhanced the sensitivity and specificity of the western blot for individual proteins. In other cases, weak blocking buffers might cause non-specific bands.

Comparison between 2% BSA, 5% NF-Milk and StartingBlock Blocking Buffer in the detection of pAKT. Method: A dilution series of 293T cell lysate starting at 10 μg/well was loaded onto Bolt 4–12% Bis-Tris-Plus gels and electrophoresed at 200 V for ~20 min. Gels were transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer Device (P0 protocol for 7 min). Immunoblotting was processed using the Bandmate Automated Western Blot Processor. Membranes were blocked with either 2% BSA (PBS), 5% Non-fat Milk (PBS), or StartingBlock Blocking Buffer. The membranes were probed with Rabbit Anti-pAKT (1:1,000, Cat. No. MA5-14916) diluted in the appropriate blocking buffer. This was followed by an incubation with HRP-conjugated Goat Anti–Rabbit IgG (1:2,500 or 0.04 µg/mL, Cat. No. 32460). Chemiluminescence detection was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate. Membranes were imaged on the iBright Imaging System.

In the detection of pAKT in 293T cell lysates, 2% BSA and StartingBlock Blocking Buffer provided the highest sensitivity. However, 2% BSA weakly blocked non-specific binding from the detection antibodies, which is seen with the non-specific banding patterns at higher total lysate load. 5% NF-Milk provided the lowest background, but at a cost to the limit of detection.

Method: A dilution series of 293T cell lysate starting at 10 μg/well was loaded onto Bolt 4–12% Bis-Tris-Plus gels and electrophoresed at 200 V for ~20 min. Gels were transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer Device (P0 protocol for 7 min). Immunoblotting was processed using the Bandmate Automated Western Blot Processor. Membranes were blocked with either 5% BSA (PBS), 5% Non-fat Milk (PBS), 1% Casein (PBS) or StartingBlock Blocking Buffer. The membranes were probed with Rabbit Anti-Hsp90 (1:5,000, Cat. No. MA1-10372) diluted in the appropriate blocking buffer. This was followed by an incubation with HRP-conjugated Goat Anti–Rabbit IgG (1:1,000 or 0.01µg/mL Cat. No. 32460). Chemiluminescence detection was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate. Membranes were imaged on the iBright Imaging System.

In the detection of highly abundant, Hsp90 in 293T cell lysates, all blocking buffers tested provided reasonable signal-to-noise ratios. 5% BSA exhibited a higher level of non-specific binding from the detection antibodies, but provided good sensitivity.

Fluorescent western blotting considerations

Particles and contaminants in blocking and wash buffers can settle on membranes and create fluorescent artifacts, so it’s important to use high-quality, filtered buffers in fluorescent western blotting. In addition, limit the use of detergents during blocking steps, as common detergents can auto-fluoresce, possibly increasing non-specific background.

Method: A dilution series of A431 cell lysate starting at 15 μg/well was loaded onto Bolt 4–12% Bis-Tris-Plus gels and electrophoresed at 200 V for ~20 min. Gels were transferred to nitrocellulose membranes using the iBlot 2 Gel Transfer Device (P0 protocol for 7 min). Membranes were blocked with either Blocker FL Blocking Buffer, 5% non-fat Milk (TBS) or another specialty blocking buffer. The membranes were probed with anti-EGFR, anti-Stat3, or anti-Ras10, diluted in the appropriate blocking buffer. This was followed by an incubation with Alexa Fluor Plus 800-conjugated Goat Anti–Rabbit IgG and Alexa Fluor Plus 680 Goat Anti-Mouse IgG. Membranes were imaged on the iBright Imaging System.


Recommended Thermo Scientific western blot blocking buffers

Select whenThermo Scientific productBlocking agentHighlightsWhen to useAvailable formats
Chemiluminescent western blotting
Optimizing a new western blot systemStartingBlock Blocking BufferSingle purified protein, serum- and biotin-free
  • Performs well with a wide range of antibodies and antibody combinations
  • Compatible with streptavidin systems
  • Blocks in less than 15 minutes
  • Current blocking buffer has high background or blocking antigen-antibody binding
  • Stripping and reprobing western blots
PBS
TBS
PBST
TBST
Traditional blockers not providing enough sensitivityBlocker CaseinPurified casein
  • High-performance replacement for homemade milk blocking buffers
  • Single-protein blocking buffer provides fewer chances of cross-reaction with assay components than serum or milk solutions
  • Targeting med-high abundant proteins or using antibodies with strong affinity
  • High background is seen with Non-fat milk blockers

PBS
TBS

Targeting phosphoproteins or need highest level of sensitivityBlocker BSAPurified BSA
  • 10% solution of high-quality BSA
  • Single purified protein provides fewer chances of cross-reaction with assay components than serum or milk solutions
  • Use when targeting phosphoproteins
  • Best to use when storing reused antibodies in blocker

10X PBS concentrate

10X TBS concentrate

Fluorescent western blotting
Performing fluorescent western detectionBlocker FL Fluorescent Blocking Buffer (10x)Single purified protein
  • Blocks excess non-specific binding sites to help reduce background fluorescence
  • Works with both nitrocellulose and low-fluorescence PVDF membranes
  • Detergent-free
  • Blocks in 15-30 minutes
Imaging and storage of dry fluorescence blots10X concentrate

Additional available blocking buffers

 Blocking agentHighlightsWhen to useAvailable formats
Pierce Clear Milk Blocking BufferClarified and stabilized milk proteins
  • High-performance replacement for homemade milk blocking buffers
  • Long shelf-life at room temperature
  • Use when high background seen with Non-fat milk
  • Fluorescent and chemiluminescent applications
Borate, pH 7.6
Fish Serum Blocking BufferSteelhead salmon serum
  • Useful in detection methods involving mammalian samples
  • Particularly effective in applications involving multiplex fluorescence imaging
  • With mammalian samples
  • Multiplexing fluorescence western blotting
PBS
SuperBlock Blocking BufferSerum- 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
Blocker BLOTTONon-fat dry milk
  • Ready-to-use 5% solution of nonfat powdered milk
  • Convenient- ready-to-use
  • More consistent product over home-made blockers
TBS
Protein-Free Blocking BufferNon-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 or provide desired probe function
PBS
TBS
PBST
TBST
Pierce Fast Blocking BufferSingle purified protein
  • Blocks in 5 minutes
  • When time is essential
TBS

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