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.
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.
|2-5% Non-fat milk||Inexpensive, contains multiple types of proteins||Contains 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 phosphoproteins||Various 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 binding||More expensive than traditional non-fat milk formulations.|
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.
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.
|Select when||Thermo Scientific product||Blocking agent||Highlights||When to use||Available formats|
|Chemiluminescent western blotting|
|Optimizing a new western blot system||StartingBlock Blocking Buffer||Single purified protein, serum- and biotin-free||PBS|
|Traditional blockers not providing enough sensitivity||Blocker Casein||Purified casein|
|Targeting phosphoproteins or need highest level of sensitivity||Blocker BSA||Purified BSA|
|Fluorescent western blotting|
|Performing fluorescent western detection||Blocker FL Fluorescent Blocking Buffer (10x)||Single purified protein||Imaging and storage of dry fluorescence blots||10X concentrate|
|Blocking agent||Highlights||When to use||Available formats|
|Pierce Clear Milk Blocking Buffer||Clarified and stabilized milk proteins||Borate, pH 7.6|
|Fish Serum Blocking Buffer||Steelhead salmon serum||PBS|
|SuperBlock Blocking Buffer||Serum- and biotin-free single purified glycoprotein||PBS|
|Blocker BLOTTO||Non-fat dry milk||TBS|
|Protein-Free Blocking Buffer||Non-protein blocking compound||PBS|
|Pierce Fast Blocking Buffer||Single purified protein||TBS|
For Research Use Only. Not for use in diagnostic procedures.