FLAG Antibody, IgG2b
Description
Western Blotting
IgG2b antibodies exhibit high sensitivity in detecting FLAG-tagged proteins. For example:
Immunoprecipitation (IP)
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Procedure: Use 10 µg/mL antibody concentration for IP in mammalian cell lysates .
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Performance: FG4R (IgG2b) effectively isolates FLAG-tagged proteins without cross-reactivity .
Immunofluorescence (IF)
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Optimal Dilution: 1:100–300 for cytoplasmic and nuclear localization studies .
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Advantage: Superior nuclear protein detection compared to anti-FLAG M2 (IgG1) in some systems .
Cross-Reactivity and Specificity
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Low Cross-Reactivity: IgG2b antibodies (e.g., PFLAGSHG) show minimal binding to endogenous proteins in bacterial and mammalian lysates .
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Comparison with Other Subclasses:
Subclass Clone Binding Dependency Key Applications IgG2b PFLAGSHG Calcium-independent WB, IP, IF IgG1 M2 Calcium-dependent Affinity chromatography IgG2a 2H8 N-terminal specificity IF, IC
Performance in Challenging Conditions
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Denaturing vs. Native Conditions:
Sensitivity and Versatility
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Sensitivity: Rat IgG mAb L5 (anti-FLAG) outperforms M2 (IgG1) in Western blot sensitivity, but IgG2b alternatives (e.g., PFLAGSHG) offer comparable performance in IP .
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Versatility: Compatible with diverse tags (e.g., C-terminal, internal) and hosts (bacteria, yeast, mammalian cells) .
Technical Considerations and Limitations
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Formulation: Avoid sodium azide in IF applications; use PBS/glycerol for long-term storage .
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Optimization: Titrate antibody dilutions for each assay to minimize background noise .
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Comparison: While IgG2b antibodies lack calcium dependency (unlike M1), they may require competitive elution with free FLAG peptide in affinity chromatography .
Product Specs
Product Science Overview
The FLAG peptide is a widely used fusion tag in molecular biology and biochemistry. It consists of eight amino acids: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK). This peptide is specifically designed for immunoaffinity chromatography, allowing for the purification and detection of recombinant fusion proteins under non-denaturing conditions .
The FLAG peptide is highly versatile and is used in various applications, including:
- Western Blotting: Detecting proteins separated by gel electrophoresis.
- Immunocytochemistry: Visualizing the localization of proteins within cells.
- Immunoprecipitation: Isolating proteins from a mixture using an antibody.
- Flow Cytometry: Analyzing the physical and chemical characteristics of cells or particles.
- Protein Purification: Isolating recombinant proteins from a mixture.
- Protein-Protein Interaction Studies: Investigating interactions between proteins.
- Cell Ultrastructure Studies: Examining the detailed structure of cells.
- Protein Localization Studies: Determining the specific location of proteins within cells .
Several monoclonal antibodies have been developed against the FLAG peptide. One such antibody, denoted as M1, binds the peptide in the presence of bivalent metal cations, preferably calcium. Elution can be achieved using chelating agents or by competitive elution with an excess of free FLAG peptide .
The FLAG peptide mouse antibody, IgG2b, is a monoclonal antibody produced by immunizing mice with the synthetic FLAG peptide coupled to keyhole limpet hemocyanin (KLH). This antibody is used in various laboratory research applications and is not intended for use as a drug, agricultural product, food additive, or household chemical .
