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In immunological tests, the accurate and reliable outcome of the assays depends significantly on the careful selection of an appropriate blocking buffer. The role of blocking buffers is crucial in minimizing non-specific binding, which is a common cause of background noise in assays such as Western blotting. Non-specific binding has the potential to obscure genuine signals, leading to inaccurate interpretations. A well-chosen blocking buffer is instrumental in improving assay sensitivity by reducing background interference, thereby enhancing the signal-to-noise ratio. This becomes especially crucial for the detection of proteins with low abundance. antibodies-online's blocking buffer formulations are specifically crafted to diminish non-specific interactions while concurrently stabilizing antibodies and antigens. This contributes to results that are more precise and enduring.

Blocking buffer principle: Blocking agents like BSA, casein, or milk proteins reduce background noise in assays by occupying non-specific binding sites on surfaces such as membranes, slides, or wells, thus preventing non-specific antibody binding and enhancing result accuracy.

To assist you in choosing the optimal blocking buffer for your assays, we have developed the following guide on selecting blocking buffers. This guide provides recommendations and precautions, ensuring the selection of the most effective blocking buffer for achieving optimal results. If you have any more questions about the buffer application, the FAQ at the bottom of the page and our customer service can both assist you.

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

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.