Rabbit IgG Isotype Control Antibody [D24D4]

Application: Reactivity:

Usage Information

Dilution

Application
IP, IHC, IF, FCM, ChIP

Reactivity

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
'-20°C (avoid freeze-thaw cycles), 2 years

Datasheet & SDS

Biological Description

Specificity
Rabbit IgG Isotype Control Antibody [D24D4] functions as an isotype control for rabbit IgG antibodies.

Clone
D24D4

Background
Rabbit IgG isotype control antibodies are immunoglobulin G class rabbit antibodies that possess the same constant region and Y-shaped structure as target-specific rabbit IgG, including paired heavy and light chains with an Fc region capable of engaging Fc receptors and complement, and Fab regions that lack specificity for antigens in the test system. The Fc portion retains the ability to bind Fcγ receptors, interact with Fc-binding proteins, and participate in non-antigen-driven associations with cellular lipids, carbohydrates, and extracellular matrix components, while the variable domains do not recognize the epitopes under investigation, resulting in a nonspecific binding profile equivalent to that of a typical rabbit primary antibody without contributing target-directed signal. Under experimental conditions, rabbit IgG isotype controls serve as negative controls to define background resulting from Fc receptor binding, weak protein–protein or protein–surface interactions, and fluorophore or enzyme conjugates, and are used at the same immunoglobulin class, subclass, and concentration as the test antibody to ensure equivalent avidity, Fc interactions, and labeling density. In flow cytometry, immunohistochemistry, and immunofluorescence, isotype control IgG binds Fc receptors on leukocytes and tissue-resident immune cells and engages nonspecific sites to the same extent as a typical rabbit primary antibody, allowing direct comparison of signal between isotype and test antibody channels and establishing a threshold that separates specific antigen-dependent staining from background. During chromatin immunoprecipitation, RIP, ELISA, or immunoprecipitation, normal rabbit IgG functions as a matrix-binding and chromatin-binding control that reports non-epitope-directed capture of nucleic acids and proteins on support surfaces under conditions identical to those used for the antigen-specific antibody. The constant-region structure of rabbit IgG enables recognition by anti-rabbit secondary antibodies and by protein A or protein G, allowing rabbit IgG isotype controls to integrate into detection and purification systems and report signal originating from secondary reagents, enzyme or fluorophore tags, or capture matrices. In multiparameter flow cytometry panels using directly conjugated rabbit antibodies, conjugated rabbit IgG isotype controls match fluorophore, isotype, and concentration, quantifying background from fluorochrome-related and Fc-mediated interactions and enabling gates and positivity thresholds to be set relative to this baseline. Across these applications, rabbit IgG isotype controls enhance assay robustness in samples with abundant Fc receptor–positive cells or high intrinsic autofluorescence by distinguishing true antigen-dependent signal from noise due to Fc–ligand interactions and nonspecific adsorption. In disease-oriented and translational studies reliant on antibody-based readouts, rabbit IgG isotype controls provide a reference for interpreting biomarker expression levels and distribution, ensuring that staining patterns attributed to pathology are not explained by altered Fc receptor expression or matrix composition alone.

Background

Rabbit IgG isotype control antibodies are immunoglobulin G class rabbit antibodies that possess the same constant region and Y-shaped structure as target-specific rabbit IgG, including paired heavy and light chains with an Fc region capable of engaging Fc receptors and complement, and Fab regions that lack specificity for antigens in the test system. The Fc portion retains the ability to bind Fcγ receptors, interact with Fc-binding proteins, and participate in non-antigen-driven associations with cellular lipids, carbohydrates, and extracellular matrix components, while the variable domains do not recognize the epitopes under investigation, resulting in a nonspecific binding profile equivalent to that of a typical rabbit primary antibody without contributing target-directed signal. Under experimental conditions, rabbit IgG isotype controls serve as negative controls to define background resulting from Fc receptor binding, weak protein–protein or protein–surface interactions, and fluorophore or enzyme conjugates, and are used at the same immunoglobulin class, subclass, and concentration as the test antibody to ensure equivalent avidity, Fc interactions, and labeling density. In flow cytometry, immunohistochemistry, and immunofluorescence, isotype control IgG binds Fc receptors on leukocytes and tissue-resident immune cells and engages nonspecific sites to the same extent as a typical rabbit primary antibody, allowing direct comparison of signal between isotype and test antibody channels and establishing a threshold that separates specific antigen-dependent staining from background. During chromatin immunoprecipitation, RIP, ELISA, or immunoprecipitation, normal rabbit IgG functions as a matrix-binding and chromatin-binding control that reports non-epitope-directed capture of nucleic acids and proteins on support surfaces under conditions identical to those used for the antigen-specific antibody. The constant-region structure of rabbit IgG enables recognition by anti-rabbit secondary antibodies and by protein A or protein G, allowing rabbit IgG isotype controls to integrate into detection and purification systems and report signal originating from secondary reagents, enzyme or fluorophore tags, or capture matrices. In multiparameter flow cytometry panels using directly conjugated rabbit antibodies, conjugated rabbit IgG isotype controls match fluorophore, isotype, and concentration, quantifying background from fluorochrome-related and Fc-mediated interactions and enabling gates and positivity thresholds to be set relative to this baseline. Across these applications, rabbit IgG isotype controls enhance assay robustness in samples with abundant Fc receptor–positive cells or high intrinsic autofluorescence by distinguishing true antigen-dependent signal from noise due to Fc–ligand interactions and nonspecific adsorption. In disease-oriented and translational studies reliant on antibody-based readouts, rabbit IgG isotype controls provide a reference for interpreting biomarker expression levels and distribution, ensuring that staining patterns attributed to pathology are not explained by altered Fc receptor expression or matrix composition alone.

References
https://pubmed.ncbi.nlm.nih.gov/28336958/ https://pubmed.ncbi.nlm.nih.gov/29263082/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Handling Instructions

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%