ELISA Blocking Buffers and Reagents

Select from a wide variety of easy-to-use and reliable blocking buffers and reagents for ELISA applications. ELISA buffers and reagents are important components to develop the best assay performance for high sensitivity, low background, and blocking non-specific binding. Find below blocking buffers, coating buffers, wash buffers, and recommended lysis buffers for success in ELISA applications. Or answer a few questions in our ELISA builder tool and receive a list of all the components you’ll need to design an ELISA that suits your unique needs.

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For a complete set of ELISA reagents, Invitrogen ELISA Buffer Kit (Cat. No. CNB0011) includes: 2 Coating Buffers (pH 7.4 and pH 9.4), Assay Buffer (Blocking Buffer), Wash Buffer, Stabilized TMB, and Stop Solution. Pair this with Matched Antibody Pair kits that come with matched antibody pairs, standards, and Streptavidin-HRP.

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Blocking buffers
Wash, coating buffers, and stop solution

Lysis buffers
AP and HRP conjugates

Recommended ELISA blocking buffers

An ELISA blocking buffer is a solution of irrelevant protein, mixture of proteins, or other compound that passively adsorbs to all remaining binding surfaces of the plate that are not occupied by the coated protein. The blocking buffer is effective if it improves the sensitivity of an assay by reducing background signal and improving the signal-to-noise ratio. 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. Select from easy-to-use and reliable blocking buffers for ELISA applications below.

Blocking buffer	Blocking agent	Highlights	When to use	Available formats (Cat. No.)
ELISA Diluent	BSA	Single purified protein provides fewer chances of cross-reaction with assay components than serum or milk solutions	Performs well with a wide range of antibodies and antibody combinations	Concentrate (DS98200)
SuperBlock Blocking Buffer	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 Compatible with streptavidin systems Blocks in less than ten minutes May be used as a protein stabilizer for drying antigen or antibody coated microplates	As a protein stabilizer when storing coated plates With streptavidin systems	PBS (37515) TBS (37535) PBST (37516) TBST (37536)
Pierce Protein-Free Blocking Buffers	Non-protein blocking compound	Helps minimize or eliminate crossreactivity 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 (37572) TBS (37570) PBST (37573) TBST (37571)

Recommended ELISA plate coating buffer, wash buffer, and stop solution

Buffer	Composition	When to use	Available formats (Cat. No.)
ELISA Plate Coating Buffer	10 mM phosphate buffer, pH 7.4	Recommended for most proteins	Concentrate (CB07100)
ELISA Plate Coating Buffer	50 mM carbonate buffer, pH 9.4	High pH aids solubility of some proteins and peptides and ensures that most proteins are unprotonated with an overall negative charge, which helps when binding to positively charged plates.	Concentrate (CB01100) Powder (28382)
ELISA Wash Buffer	Tris-buffered saline with 0.05% Tween 20		Concentrate (28360) Powder (28379)
ELISA Wash Buffer	Phosphate-buffered saline with 0.05% Tween 20		Concentrate (28352) Powder (00-0400-46)
ELISA Diluent	Phosphate-buffered solution with BSA	ELISA Diluent is designed to be used as (1) a blocking reagent for ELISA plates, (2) a diluent for ELISA standards and samples, (3) as a diluent for the detector antibody, and (4) as a diluent for the HRP conjugate	Concentrate (00-4202-56)
Stop Solution	0.16 M sulfuric acid	Use as a stop solution when horseradish peroxidase and TMB substrate solution are used	Ready-to-use (N600)

Recommended cell lysis buffers

Buffer	Composition	When to use	Available formats (Cat. No.)
NP40 Cell Lysis Buffer	50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na₃VO₄, 1% NP40, 0.02% NaN₃	Most popular and recommended for lysis of cell membrane and cytoplasmic proteins to measure intracellular proteins using ELISA	Ready-to-use (FNN0021)
RIPA Cell Lysis Buffer	10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate	Need a harsher buffer than NP40 or when nuclear disruption is also needed	Ready-to-use (FNN0011)
Tissue Extraction Reagent I	50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 2 mM Na₃VO₄, 1 mM NaF, 20 mM Na₄P₂O₇, 0.02% NaN₃, and detergent	For total protein extraction from tissue samples or alternate formulation for total protein lysis if NP40 or RIPA is not sufficient	Ready-to-use (FNN0071)

AP and HRP conjugates

Select from a wide variety of conjugates and detection probes for use in direct or indirect sandwich ELISA applications. Below are recommended biotin-binding probes including streptavidin and NeutrAvidin protein. Each protein binds four biotins per molecule with high affinity and selectivity. Streptavidin is most commonly used—it is non-glycosylated and exhibits low levels of nonspecific binding. NeutrAvidin has been processed to remove the carbohydrate and lower its isoelectric point, resulting in reduced nonspecific background staining.

For directly conjugated primary and secondary antibodies, search antibody enzyme conjugates

Recommended HRP Conjugates and AP Conjugates for ELISA applications

Conjugate	Enzyme	Recommended concentration*	Benefits, when to use	Available formats (Cat. No.)
HRP- Streptavidin	HRP	0.1 µg/mL	Most popular	2 mg (21124) 1 mg (21126) 5 mg (21127)
Poly-HRP Streptavidin	HPR	1:2,000 - 1:20,000	Use when exhibiting weak signal with typical HRP conjugates. Poly increases signal amplification and improves upon the signal provided by typical (non-Poly) streptavidin-HRP conjugates	0.25 mL (N200)
HRP-NeutrAvidin	HRP	0.1 µg/mL	Specially prepared form of avidin that decreases background in biotin-binding	2 mg (31001)
HRP-NeutrAvidin, High Sensitivity	HRP	1:8,000 - 1:40,000	Detection of low levels of target without background; provides signal amplification like poly-HRP conjugates	0.5 mL (31030)
AP-Streptavidin	AP	0.1 µg/mL		1 mg (21324)
AP-NeutrAvidin	AP		Specially prepared form of avidin that decreases background in biotin-binding	2 mg (31002)

* The optimal dilution must be determined empirically for each specific application. Prepare dilutions immediately before use.

Search all available Avidin, Streptavidin, and Biotin conjugates

Protocols and technical guides

Blocking buffers

ELISA lysis buffers

ELISA Buffers