Blocking Buffers
Julian Pampel
In immunological assays, selecting the right blocking buffer is crucial for accurate and reliable results. Blocking buffers play an essential role in reducing non-specific binding, a common source of background noise in assays like Western blotting. This non-specific binding can obscure true signals, leading to inaccurate interpretations. A well-chosen blocking buffer enhances assay sensitivity by minimizing background interference, thereby improving the signal-to-noise ratio. This is vital for detecting low-abundance proteins. Rockland's blocking buffer formulations are designed to reduce nonspecific interactions while stabilizing antibodies and antigens, contributing to more precise and durable results.
To aid in selecting the ideal blocking buffer for your assays, we have created the following guide to blocking buffer selection. This guide offers recommendations and precautions, ensuring you choose the most effective blocking buffer for optimal results. Importantly, these buffers are also utilized in-house at Rockland, reflecting our commitment to quality and reliability in both our products and research.
Blocking Buffer Selection Guide
| Product | Recommended Use | Application Note | Precautions |
|---|---|---|---|
| Blocking Buffer for Fluorescent Western Blotting | Fluorescent WB, ELISA, IF, IHC, Microarray, Multiplex | Ready-to-use solution. Designed for use in fluorescent Western blotting but also useful in other immunoassays with fluorescent labels. | Contains thimerosal, for a thimerosal-free option please see Blocking Buffer (10X) for Fluorescent Western Blotting (Thimerosal Free). |
| Blocking Buffer (2X) for Fluorescent Western Blotting | Fluorescent WB, ELISA, IF, IHC, Microarray, Multiplex | 2X concentrated solution. Designed for use in fluorescent Western blotting but also useful in other immunoassays with fluorescent labels. | Contains thimerosal, for a thimerosal-free option please see Blocking Buffer (10X) for Fluorescent Western Blotting (Thimerosal Free). |
| Blocking Buffer for Fluorescent Western Blotting (Thimerosal Free) 10-Pack | Fluorescent WB, ELISA, IF, IHC, Microarray, Multiplex | 10-pack of ready-to-use solution. Thimerosal-free blocking buffers are often preferred in facilities where safety and environmental impact are significant considerations. | |
| Blocking Buffer (10X) for Fluorescent Western Blotting (Thimerosal Free) | Fluorescent WB, ELISA, IF, IHC, Microarray, Multiplex | 10X concentrated solution. Thimerosal-free blocking buffers are often preferred in facilities where safety and environmental impact are significant considerations. | |
| Normal Goat Serum (NGS) | WB, ELISA, IF, IHC | For use in immunoassays where the primary antibody was produced in goat. | |
| Normal Horse Serum (NHS) | WB, ELISA, IF, IHC | For use in immunoassays where the primary antibody was produced in horse. | |
| Normal Mouse Serum (NMS) | WB, ELISA, IF, IHC | For use in immunoassays where the primary antibody was produced in mouse. | |
| Normal Rabbit Serum (NRS) | WB, ELISA, IF, IHC | For use in immunoassays where the primary antibody was produced in rabbit. | |
| Normal Rat Serum (NRS) | WB, ELISA, IF, IHC | For use in immunoassays where the primary antibody was produced in rat. | |
| Normal Sheep Serum (NSS) | WB, ELISA, IF, IHC | For use in immunoassays where the primary antibody was produced in sheep. | |
| Bovine Serum Albumin 30% Solution | ELISA, WB, IHC | BSA blocking solutions are preferred with biotin and AP antibody labels. | |
| Bovine Serum Albumin - Fraction V | ELISA, WB, IHC | BSA blocking solutions are preferred with biotin and AP antibody labels. | Since BSA is notoriously difficult to dissolve, you can save time using our Bovine Serum Albumin 30% Solution and dilute further. |
| ELISA Microwell Blocking Buffer with Stabilizer | ELISA | Designed to block microwells coated with antigens, antibodies or other ligands and stabilize the plates for drying. | |
| Blotto Immunoanalytical Grade (Non-Fat Dry Milk) | WB, ELISA | Immunoanalytical grade non-fat dry milk. |
Not suitable for use with biotin-avidin detection due to inherent biotin content. |
| Blotto A Pre-Mixed | WB, ELISA | General purpose blocking agent. | |
| Blotto B Pre-Mixed | WB, ELISA | General purpose blocking agent when phospho-specific antibodies are used. | |
| 10X BBS Fish Gel Concentrate | WB, IHC, IF, Multiplex | Fish gel is less likely to cross-react with antibodies of mammalian origin than conventional blocking reagents such as non-fat dry milk and BSA. | |
| 10X PBS Fish Gel Concentrate | WB, IHC, IF, Multiplex | Fish gel is less likely to cross-react with antibodies of mammalian origin than conventional blocking reagents such as non-fat dry milk and BSA. | For AP antibody labels use 10X TBS Fish Gel Concentrate or 10X BBS Fish Gel Concentrate buffers instead. |
| 10X TBS Fish Gel Concentrate | WB, IHC, IF, Multiplex | Fish gel is less likely to cross-react with antibodies of mammalian origin than conventional blocking reagents such as non-fat dry milk and BSA. | |
| 1X PBS, pH 7.2 Buffer with 1% Casein | WB | Blocking buffers containing casein may provide lower backgrounds than buffers containing non-fat milk or BSA. Casein is recommended for applications using biotin-avidin complexes. | For AP antibody labels use 1X TBS, pH 7.8 Buffer with 1% Casein instead. |
| 1X TBS, pH 7.8 Buffer with 1% Casein | WB | Blocking buffers containing casein may provide lower backgrounds than buffers containing non-fat milk or BSA. Casein is recommended for applications using biotin-avidin complexes. | |
| Blocking Buffer Sampler Kit | Fluorescent WB, WB, ELISA, IF, IHC, Microarray, Multiplex | Set of different blocking buffers for all sorts of immunoassays. |
Blocking Buffer FAQ
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What is the main purpose of a blocking buffer in immunodetection assays?
The main purpose of a blocking buffer is to prevent non-specific binding of antibodies to the assay surface, thereby reducing background noise and increasing the specificity and accuracy of the assay results. -
What are common components of a blocking buffer?
Common components of blocking buffers include proteins like bovine serum albumin (BSA), casein, or non-fat milk proteins. These substances effectively cover non-specific binding sites on the assay surface. -
How does a blocking buffer contribute to the accuracy of an ELISA test?
In an ELISA test, a blocking buffer minimizes non-specific binding of antibodies to the well surface, reducing background noise. This enhances the signal-to-noise ratio, leading to more accurate and reliable quantification of the target antigen. -
Can blocking buffers affect antibody-antigen binding?
No, when used correctly, blocking buffers do not interfere with the specific binding of antibodies to their target antigens. Instead, they prevent antibodies from binding to irrelevant sites, thus ensuring that the signal observed is due to specific antigen-antibody interactions. -
Is it necessary to use a blocking buffer in all types of immunoassays?
While highly recommended, the necessity of a blocking buffer can depend on the specific assay and its sensitivity to background noise. In assays where specificity and low background are critical, such as in highly sensitive ELISAs or Western blots, the use of a blocking buffer is essential. -
Are there alternatives to protein-based blocking buffers?
Yes, alternatives to protein-based blocking buffers include synthetic polymers and detergents. These can be used in cases where protein-based buffers might cross-react with the antibodies or antigens in the assay. -
How long should the blocking step be in an immunoassay?
The duration of the blocking step can vary depending on the assay, but it typically ranges from 30 minutes to several hours. The optimal blocking time may need to be determined empirically for each specific assay.
