Anti-Mouse CD32/CD16 [Clone 2.4G2] — Purified in vivo GOLD™ Functional Grade

Anti-Mouse CD32/CD16 [Clone 2.4G2] — Purified in vivo GOLD™ Functional Grade

Product No.: C381

[product_table name="All Top" skus="C381"]

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Clone
2.4G2
Target
CD32/CD16
Formats AvailableView All
Product Type
Monoclonal Antibody
Alternate Names
Fcγ R III/II, Ly-17
Isotype
Rat IgG2b
Applications
B
,
FA
,
FC
,
IHC FF
,
in vivo
,
IP
,
PhenoCycler®
,
WB

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Data

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Antibody Details

Product Details

Reactive Species
Mouse
Host Species
Rat
Recommended Isotype Controls
Recommended Dilution Buffer
Immunogen
Sorted pre-B cells
Product Concentration
≥ 5.0 mg/ml
Endotoxin Level
< 1.0 EU/mg as determined by the LAL method
Purity
≥95% monomer by analytical SEC
>95% by SDS Page
Formulation
This monoclonal antibody is aseptically packaged and formulated in 0.01 M phosphate buffered saline (150 mM NaCl) PBS pH 7.2 - 7.4 with no carrier protein, potassium, calcium or preservatives added. Due to inherent biochemical properties of antibodies, certain products may be prone to precipitation over time. Precipitation may be removed by aseptic centrifugation and/or filtration.
Product Preparation
Functional grade preclinical antibodies are manufactured in an animal free facility using in vitro cell culture techniques and are purified by a multi-step process including the use of protein A or G to assure extremely low levels of endotoxins, leachable protein A or aggregates.
Storage and Handling
Functional grade preclinical antibodies may be stored sterile as received at 2-8°C for up to one month. For longer term storage, aseptically aliquot in working volumes without diluting and store at ≤ -70°C. Avoid Repeated Freeze Thaw Cycles.
Country of Origin
USA
Shipping
Next Day 2-8°C
Applications and Recommended Usage?
Quality Tested by Leinco
FC The suggested concentration for this 2.4G2 antibody for staining cells in flow cytometry is ≤ 1 μg per 106 cells in a volume of 100 μl. Titration of the reagent is recommended for optimal performance for each application.
WB The suggested concentration for this 2.4G2 antibody for use in western blotting is 1-10 μg/ml.
Additional Applications Reported In Literature ?
CODEX®
Additional Reported Applications For Relevant Conjugates ?
B
IP
For specific conjugates of this clone, review literature for suggested application details.
Each investigator should determine their own optimal working dilution for specific applications. See directions on lot specific datasheets, as information may periodically change.

Description

Description

Specificity
Clone 2.4G2 recognizes the FcγIII and FcγII receptors.
Background
CD16 is expressed in two forms: CD16a and CD16b. CD16a (FcγRIIIA) is a 50-65 kD polypeptide-anchored transmembrane protein. CD16b (FcγRIIIB) is a 48 kD GPI-anchored protein whose extracellular domain is over 95% homologous to that of CD16a. CD16 regulates both phagocytosis and antibody-dependent cell-mediated cytotoxicity. It has been reported that CD16 is involved in Natural Killer Cell activation and plays a role in signal transduction. The receptors, CD32 (FcγRIII) and CD16 (FcγRII), are 40-60 kD and bind antibody-antigen immune complexes and mediate adaptive immune responses.
Antigen Distribution
These receptors are present on B cells, monocyte/macrophages, NK cells, neutrophils, mast cells and dendritic cells.
Ligand/Receptor
IgG
Function
Low affinity receptors for IgG
PubMed
NCBI Gene Bank ID
Research Area
Immunology
.
Innate Immunity

Leinco Antibody Advisor

Powered by AI: AI is experimental and still learning how to provide the best assistance. It may occasionally generate incorrect or incomplete responses. Please do not rely solely on its recommendations when making purchasing decisions or designing experiments.

Clone 2.4G2 is utilized in in vivo mouse studies primarily as an Fc receptor blocking antibody to prevent non-specific binding and interference from endogenous antibodies during experimental procedures.

Primary Mechanism and Purpose

The 2.4G2 antibody works by blocking the interaction between IgG antibodies and CD16 (Fc?RIII) or CD32 (Fc?RII) receptors on immune cells. These receptors are expressed on various immune cell types including B cells, monocytes, macrophages, NK cells, neutrophils, granulocytes, mast cells, and dendritic cells. By occupying these Fc receptor binding sites, the 2.4G2 antibody prevents non-specific binding of experimental antibodies that could interfere with study results.

Specialized In Vivo Formulations

For in vivo applications, specialized formulations of the 2.4G2 antibody have been developed with specific characteristics that make them suitable for use in living mice:

Low Endotoxin Content: The in vivo grade formulations contain endotoxin levels of ?0.01 EU/?g or <1 EU per 1 mg, as determined by the Limulus amebocyte lysate (LAL) test. This low endotoxin level is crucial to prevent inflammatory responses that could confound experimental results.

Azide-Free Formulation: Unlike some laboratory antibodies that contain sodium azide as a preservative, the in vivo grade 2.4G2 is formulated without azide, making it safe for injection into living animals.

Modified Variants for In Vivo Use

Several engineered versions of the 2.4G2 antibody have been developed specifically for enhanced in vivo performance:

LALA-PG Mutation: Some versions contain LALA-PG mutations in the Fc fragment, which render the antibody unable to bind to endogenous Fc? receptors. This modification prevents the blocking antibody itself from causing unwanted immune activation while still allowing it to block the target receptors.

Species Switching: Chimeric versions have been created where the variable domains remain identical to the original 2.4G2, but the constant regions are switched from rat IgG2b to mouse IgG2a. This modification can reduce immunogenicity in mouse models.

Applications in Mouse Studies

In vivo applications of 2.4G2 include blocking Fc receptors during studies investigating antibody-mediated immune responses, preventing interference in flow cytometry analysis of tissues harvested from treated mice, and neutralizing endogenous Fc receptor activity in functional assays studying biological pathways affected by CD16 and CD32 proteins. The antibody is also used in bioanalytical pharmacokinetic (PK) and anti-drug antibody (ADA) assays in mouse models.

The careful formulation and engineering of these in vivo grade 2.4G2 antibodies ensures they can be safely administered to mice while effectively blocking Fc receptors without causing adverse reactions or confounding experimental results.

The correct storage temperature for sterile packaged clone 2.4G2 (anti-mouse CD16/CD32 monoclonal antibody) is 2–8°C for short-term (up to 1 month) and -20°C to -70°C for long-term storage (up to 12 months). It is important to avoid repeated freeze-thaw cycles and use a manual defrost freezer for long-term storage.

Essential context:

  • Upon receipt, store immediately at the recommended temperature; products are often shipped on ice packs, which is for transit, not storage.
  • For use within 1 month, 2–8°C (standard refrigerator) is adequate.
  • For periods longer than 1 month, -20°C to -70°C preserves antibody stability and activity for up to 12 months.

Additional information:

  • Protect the antibody from light, especially if it is PE-conjugated.
  • Avoid freeze-thaw cycles to prevent degradation; aliquoting is recommended for long-term storage.
  • Storage conditions apply equally to both basic and conjugated forms, unless stated otherwise in product documentation.
  • These guidelines reflect widely accepted manufacturer recommendations for clone 2.4G2 antibodies.

Commonly Used Antibodies and Proteins with 2.4G2 in the Literature

The 2.4G2 monoclonal antibody is best known for its role in blocking mouse CD16 and CD32 (Fc?RIII and Fc?RII), preventing non-specific binding of the Fc portion of immunoglobulins during immunoassays such as flow cytometry and immunofluorescence. However, when used in research, 2.4G2 is not typically the primary immune marker; rather, it is employed as a blocking reagent to improve the specificity of other antibodies targeting cell surface markers. Below are the main categories of antibodies and proteins commonly used in concert with 2.4G2 in the literature.

Common Antibody Partners in Multiparameter Analysis

In mouse immunology and cell phenotyping studies, 2.4G2 is used alongside a variety of fluorescently labeled antibodies that target specific leukocyte subsets or activation markers. Examples from the literature include:

Target ProteinClone(s)ApplicationsNotes
B220 (CD45R)RA3-6B2B cell identificationOften FITC or APC conjugated.
CD3e145-2C11T cell identificationFITC, BV510, or AlexaFluor 647 conjugated.
CD11bM1/70Myeloid cell identificationPerCP-Cy5.5 conjugated.
CD196D5B cell identificationBV510 conjugated.
CD4530-F11Pan-leukocyte markerAPC-Cy7, APC-Fire750 conjugated.
CD49b (DX5)DX5NK cell, platelet identificationAPC conjugated.
CD62LMEL-14Lymphocyte activationPE-Cy7 conjugated.
Gr1 (Ly6G/Ly6C)RB6-8C5, 1A8Granulocyte, monocyte identificationAPC, AlexaFluor 647, BV510, FITC conjugated.
IgER35-72Basophil/mast cell identificationFITC conjugated.
NK1.1PK136NK cell identificationFITC conjugated, supplied by multiple vendors.
TCR?H57-597T cell identificationFITC conjugated.

These antibodies are routinely used in panels for comprehensive immune cell profiling, where 2.4G2 is added as a blocking reagent to minimize non-specific Fc receptor-mediated staining before applying these primary antibodies.

Blocking with 2.4G2 in Functional Assays

  • Primary Antibodies: Following Fc receptor blockade with 2.4G2, primary antibodies against specific cell surface markers (e.g., CD4, CD8, B220, CD11b, Gr1, NK1.1) are used to identify and sort cell populations.
  • Secondary Antibodies: If a secondary antibody step is used (e.g., for indirect staining), it must not be anti-rat IgG2b, as 2.4G2 is a rat antibody and would otherwise be detected by such a secondary antibody.
  • Functional Assays: In addition to flow cytometry, 2.4G2 is used in neutralization, pharmacokinetic (PK), and anti-drug antibody (ADA) assays to block Fc-mediated interactions that could confound results.

Other Proteins and Controls

  • Isotype Controls: Mouse IgG2a (the isotype of 2.4G2) or irrelevant isotype-matched antibodies are often used as negative controls to confirm specificity in staining experiments.
  • Recombinant Fc Proteins: Sometimes, purified Fc fragments or recombinant Fc proteins are used in parallel with 2.4G2 to confirm that blocking is effective and to study Fc receptor biology.
  • Complement and Immune Complexes: In functional studies of phagocytosis or ADCC, 2.4G2 may be used to block Fc receptors before adding complement or immune complexes to assess receptor-specific contributions.

Summary Table of Common Antibodies Used with 2.4G2

ApplicationExample Antibodies/ProteinsPurpose
Cell Surface MarkersB220, CD3, CD19, CD11b, Gr1, NK1.1Immune cell phenotyping
Isotype ControlsMouse IgG2a, irrelevant IgGSpecificity controls
Secondary AntibodiesAnti-IgG (not anti-rat IgG2b)Indirect detection
Functional AssaysFc fragments, complement proteinsFc receptor biology/blockade validation

Conclusion

In the literature, 2.4G2 is most frequently used as a blocking reagent in combination with a wide array of cell surface marker antibodies (e.g., B220, CD3, CD19, CD11b, Gr1, NK1.1) for multiparameter flow cytometry and immunofluorescence in mouse samples. Appropriate isotype controls and careful selection of secondary antibodies (avoiding anti-rat IgG2b) are also standard practice. These combinations enable precise identification and analysis of immune cell subsets by minimizing non-specific staining through Fc receptor blockade.

Clone 2.4G2 is a monoclonal antibody widely used in immunology research, especially in mouse models, and is most frequently cited for its ability to block or detect Fc gamma receptor II (CD32, Fc?RII) and Fc gamma receptor III (CD16, Fc?RIII). Key findings and scientific uses from the literature include:

  • Widely Used as an Fc Blocker: The primary use of clone 2.4G2 is to block Fc?RII and Fc?RIII on mouse immune cells during experiments (such as flow cytometry and immunofluorescence) to prevent non-specific binding of antibodies via their Fc region. This increases the specificity of staining for target antigens.

  • Specificity and Additional Binding: While 2.4G2 is specific for CD16 (Fc?RIII) and CD32 (Fc?RII), it has also been reported to exhibit non-specific binding to Fc?RI (CD64) via its own Fc region when the antibody is already bound to Fc?RII or Fc?RIII, making careful interpretation necessary in certain analyses.

  • Blocking in Quantitative and Functional Analyses: Studies frequently employ 2.4G2 for blocking during flow cytometry, especially when quantifying or characterizing Fc? receptor expression or to avoid artifacts caused by Fc-mediated binding. The antibody is also used to block Fc receptors in vivo in animal models.

  • Cell Types Affected: CD16 and CD32 are expressed on a variety of mouse immune cells, including B cells, NK cells, macrophages, granulocytes, mast cells, and dendritic cells.

  • Use in Functional Studies: In addition to flow cytometry, clone 2.4G2 has been used in research to study functions regulated by Fc?RII and Fc?RIII, such as phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC), as well as in analyzing Natural Killer (NK) cell function.

  • Critical Considerations:

    • Use in Immunoassays: When blocking with 2.4G2, care must be taken in subsequent detection steps, specifically avoiding secondary reagents that are anti-rat IgG2b, which may cross-react.
    • Limitations: 2.4G2 does not block all Fc gamma receptors; for example, Fc?RIV is not effectively blocked, so additional reagents (like clone 9E9) might be needed in experiments studying multiple Fc receptor types.
  • Benchmark in Fc Receptor Research: The 2.4G2 clone has been cited in hundreds of publications and is considered a standard tool for Fc receptor research in immunology.

In summary, the main scientific contribution of clone 2.4G2 is its reliable and efficient blocking of Fc?RII and Fc?RIII, which underpins much of mouse immunology research by allowing for more specific analysis of immune cell markers and functions.

References & Citations

1.) Titas, J. A. et al. (1982) J. Immunol. 133:556
2.) Rodewald, H. et al. (1992) Cell 69:139
3.) Skyberg, J. A. et al. (2020) Infection and Immunity. 88: 5
4.) Forte et al. (2020) Cell Reports. 30:3149–3163 Journal Link
5.) Forte, E. et al. (2020) Cell Reports 30(9):3149-3163.e6 Journal Link
B
FA
Flow Cytometry
IHC FF
in vivo Protocol
Immunoprecipitation Protocol
PhenoCycler®
General Western Blot Protocol

Certificate of Analysis

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Disclaimer AlertProducts are for research use only. Not for use in diagnostic or therapeutic procedures.