RGT2 Antibody
Description
Overview of RAG2 Antibody
RAG2 is a core component of the RAG complex (RAG1/RAG2), which mediates DNA cleavage during V(D)J recombination in developing lymphocytes. This process is essential for generating diverse antigen receptors in B and T cells .
The RAG2 Antibody (11825-1-AP) from Proteintech is a polyclonal rabbit IgG antibody validated for use in Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF/ICC), immunoprecipitation (IP), and ELISA .
Host and Reactivity
| Property | Details |
|---|---|
| Host Species | Rabbit |
| Isotype | IgG |
| Reactivity | Human, Mouse, Rat |
| Tested Applications | WB, IHC, IF/ICC, IP, ELISA |
Immunogen and Molecular Data
Applications and Dilutions
| Application | Recommended Dilution | Validated Samples |
|---|---|---|
| Western Blot (WB) | 1:200 – 1:1000 | A375 cells, mouse thymus tissue |
| Immunoprecipitation (IP) | 0.5–4.0 µg/1–3 mg lysate | A375 cells |
| IHC | 1:20 – 1:200 | Human lymphoma tissue |
| IF/ICC | 1:10 – 1:100 | HeLa cells |
Key Findings
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RAG2 is non-catalytic but essential for recruiting the RAG complex to chromatin via H3K4me3 interactions .
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Hypomorphic RAG2 mutations can lead to immune dysregulation, including hyper-IgM phenotypes and self-reactive B cell expansion, though clinical autoimmunity is rare .
Clinical and Research Relevance
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Role in Immune Disorders: Hypomorphic RAG2 variants are linked to combined immunodeficiency (CID) and atypical presentations like late-onset hyper-IgM syndrome .
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Therapeutic Targets: The RAG complex is studied for modulating immune receptor diversity in gene therapy and autoimmune disease research .
Comparison of Antibody Performance
Protocol Recommendations
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Antigen Retrieval: For IHC, use TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
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Storage: Stable at -20°C for 1 year; avoid freeze-thaw cycles .
Limitations and Considerations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Constitutive mutations within RGT2 affect evolutionarily conserved amino acids in the transmembrane spanning regions of the protein. These mutations cause RGT2 to adopt inward-facing or occluded conformations, effectively generating the glucose signal. PMID: 29954842
- The RGT2-1 variant promotes ubiquitination and subsequent degradation of Mth1 and Std1, irrespective of the presence of glucose. PMID: 17586499
KEGG: sce:YDL138W
STRING: 4932.YDL138W
Q&A
What is TGF-beta RII Antibody and what is its target?
TGF-beta RII antibody specifically recognizes and binds to TGF-beta receptor type II (TGF-beta RII), a membrane-bound serine/threonine kinase that plays a crucial role in TGF-beta signaling. This receptor binds TGF-beta 1 and TGF-beta 3 with high affinity, while binding TGF-beta 2 with much lower affinity. The receptor is approximately 70-85 kDa in size and is expressed in various cell types. The antibody serves as a valuable tool for detecting and studying this receptor in research applications .
What are the primary applications of TGF-beta RII Antibody in research?
TGF-beta RII antibody has been validated for multiple research applications including:
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Western blotting - For detection of TGF-beta RII in protein lysates
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Flow cytometry - For cell surface or intracellular detection of the receptor
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Direct ELISA - For quantitative detection of the target protein
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Immunocytochemistry - For visualization of receptor localization in cells
These applications make the antibody versatile for investigating TGF-beta signaling in various experimental contexts, from protein expression analysis to cellular localization studies .
What species reactivity has been confirmed for TGF-beta RII Antibody?
The antibody has been specifically developed against mouse TGF-beta RII, with validated reactivity in mouse systems. In direct ELISAs, less than 10% cross-reactivity has been observed with recombinant human TGF-beta RII, indicating good species specificity while maintaining some cross-species reactivity that may be useful in comparative studies .
How can TGF-beta RII Antibody be used to investigate TGF-beta signaling in immune regulation?
TGF-beta RII antibody has been valuable in studying immune regulation, particularly in examining age-dependent changes in immune function. Research has shown that intrinsic TGF-beta signaling contributes to age-dependent CD8+ T cell polyfunctionality attrition, as demonstrated in studies using this antibody for flow cytometry applications. The antibody can help researchers detect changes in receptor expression levels in different immune cell populations, allowing for correlation with functional changes in immune responses .
For investigating immune regulation, researchers should:
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Use multiparameter flow cytometry with proper controls to identify specific immune cell subsets
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Compare TGF-beta RII expression across different immune cell populations
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Correlate receptor expression with functional assays such as cytokine production
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Consider age-related variables when studying TGF-beta signaling in immune cells
What are the technical considerations for using TGF-beta RII Antibody in multi-parameter flow cytometry experiments?
When designing multi-parameter flow cytometry experiments with TGF-beta RII antibody, researchers should consider:
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Panel design: Ensure that fluorophore selection minimizes spectral overlap with other markers
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Titration: Determine optimal antibody concentration through titration experiments
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Controls: Include appropriate isotype controls (such as catalog # AB-108-C) to establish specificity
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Compensation: Properly compensate for spectral overlap between fluorophores
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Sample preparation: Follow validated protocols for cell preparation, such as those for staining membrane-associated proteins
Published studies have successfully used the antibody in flow cytometry to analyze TGF-beta RII expression in mouse splenocytes, demonstrating its compatibility with multi-parameter analysis .
How can researchers address discrepancies between TGF-beta RII Antibody results and other methodologies?
When encountering discrepancies between results obtained with TGF-beta RII antibody and alternative methods:
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Validate antibody specificity: Confirm specificity using knockout/knockdown controls
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Compare detection limits: Different methods may have varying sensitivity thresholds
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Consider post-translational modifications: Some antibodies may detect specific forms of the receptor
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Evaluate experimental conditions: Buffer compositions, fixation methods, and epitope accessibility can affect results
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Cross-validate with multiple antibodies: Use antibodies targeting different epitopes of TGF-beta RII
A systematic approach comparing multiple detection methods provides the most robust validation of experimental findings related to TGF-beta RII expression and function.
What are the recommended protocols for using TGF-beta RII Antibody in Western blot applications?
For optimal Western blot results with TGF-beta RII antibody:
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Sample preparation: Prepare lysates in appropriate buffer (Immunoblot Buffer Group 8 has been validated)
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Reduction conditions: Perform experiments under reducing conditions
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Protein amount: Load adequate protein (10-20 μg per lane recommended)
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Antibody dilution: Use at 2 μg/mL concentration
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Detection system: Use HRP-conjugated secondary antibody (Anti-Goat IgG)
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Expected band size: Look for specific band at approximately 75 kDa
Scientific data confirms the detection of mouse TGF-beta RII in mouse brain tissue lysates using this methodology, with the specific band appearing at approximately 75 kDa .
How should TGF-beta RII Antibody be stored and handled to maintain optimal activity?
| Storage Condition | Duration | Temperature | Notes |
|---|---|---|---|
| As supplied | 12 months | -20 to -70°C | Avoid freeze-thaw cycles |
| After reconstitution | 1 month | 2 to 8°C | Keep under sterile conditions |
| After reconstitution | 6 months | -20 to -70°C | Keep under sterile conditions |
For optimal performance:
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Use a manual defrost freezer
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Avoid repeated freeze-thaw cycles
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Reconstitute lyophilized antibody carefully according to manufacturer instructions
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Aliquot reconstituted antibody to minimize freeze-thaw cycles
What controls should be included when using TGF-beta RII Antibody in experimental procedures?
When using TGF-beta RII antibody, include the following controls:
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Negative controls:
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Isotype control antibody (e.g., catalog # AB-108-C for flow cytometry)
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Samples known to be negative for TGF-beta RII
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Secondary antibody-only controls
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Positive controls:
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Tissues/cells known to express TGF-beta RII (e.g., mouse brain tissue, splenocytes)
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Recombinant TGF-beta RII protein
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Specificity controls:
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Blocking with recombinant protein
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Competitive inhibition assays
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Knockout/knockdown samples when available
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Published studies have utilized appropriate controls when implementing this antibody in flow cytometry and Western blot applications, confirming its specificity for TGF-beta RII .
How can TGF-beta RII Antibody be incorporated into studies investigating receptor trafficking?
For studying TGF-beta RII trafficking:
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Time-course experiments: Treat cells with TGF-beta ligands and measure receptor internalization at various timepoints
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Co-localization studies: Combine TGF-beta RII antibody with markers for different cellular compartments (endosomes, lysosomes, etc.)
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Surface vs. intracellular staining: Compare cell surface staining with permeabilized cell staining to assess internalization
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Pulse-chase experiments: Label surface receptors, allow internalization, and track receptor fate
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Inhibitor studies: Use endocytosis inhibitors to block specific trafficking pathways
These approaches can help elucidate the dynamics of TGF-beta RII trafficking in response to various stimuli and in different cell types.
What experimental approaches can be used to study TGF-beta RII-mediated signaling using this antibody?
To investigate TGF-beta RII-mediated signaling:
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Receptor activation studies: Treat cells with TGF-beta ligands and assess downstream signaling
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Phosphorylation analysis: Combine with phospho-specific antibodies against Smad proteins
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Co-immunoprecipitation: Use the antibody to pull down receptor complexes and identify binding partners
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Reporter assays: Combine receptor detection with functional readouts of TGF-beta signaling
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Inhibition studies: Use in conjunction with TGF-beta pathway inhibitors
Evidence from published research demonstrates the utility of this approach, as seen in studies examining augmented cytoplasmic Smad4 and acceleration of TGF-beta1 signaling in renal tubulointerstitial cells, where TGF-beta RII antibody helped elucidate signaling mechanisms .
How should researchers interpret variations in signal intensity when using TGF-beta RII Antibody across different cell types?
When analyzing variations in TGF-beta RII staining intensity:
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Baseline expression levels: Different cell types naturally express varying levels of TGF-beta RII
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Receptor accessibility: Cell-specific membrane composition may affect antibody binding
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Co-receptor expression: Consider the expression of TGF-beta RI and other co-receptors
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Activation state: Cells in different activation states may show altered receptor expression
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Quantification methods: Use appropriate quantification (MFI for flow cytometry, densitometry for Western blots)
Research has shown differential expression of TGF-beta RII across cell types, including variations between immune cell subsets and differences in expression patterns in normal versus diseased tissues .
What are common sources of false positives or false negatives when using TGF-beta RII Antibody?
| Source of Error | Potential Solutions |
|---|---|
| Insufficient blocking | Optimize blocking conditions using BSA or serum |
| Cross-reactivity | Validate specificity with proper controls |
| Epitope masking | Try alternative fixation/permeabilization methods |
| Antibody degradation | Follow storage recommendations, use fresh aliquots |
| Signal saturation | Titrate antibody, adjust exposure/detection settings |
| Low target expression | Increase sample amount, use more sensitive detection |
| High background | Increase washing steps, decrease antibody concentration |
| Non-specific binding | Include appropriate blocking agents (e.g., Fc block) |
Researchers should systematically evaluate these potential sources of error when troubleshooting unexpected results with TGF-beta RII antibody.
How has TGF-beta RII Antibody been used in cancer research models?
TGF-beta RII antibody has been employed in several cancer research applications:
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Tumor microenvironment studies: Investigating TGF-beta signaling in tumor-infiltrating immune cells
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Metastasis research: Examining the role of TGF-beta in epithelial-mesenchymal transition
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Therapeutic targeting: Evaluating blockade of TGF-beta signaling as a therapeutic strategy
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Biomarker development: Assessing TGF-beta RII expression as a potential prognostic marker
Research has utilized this antibody to study bone morphogenetic protein and activin membrane-bound inhibitor overexpression, demonstrating inhibition of gastric tumor cell invasion via the TGF-beta/epithelial-mesenchymal transition signaling pathway .
Additionally, the antibody has been valuable in developing syngeneic murine cell lines for immunocompetent orthotopic lung cancer models, helping researchers establish and characterize these important research tools .
What is the role of TGF-beta RII Antibody in studying age-related immune dysfunction?
TGF-beta RII antibody has provided critical insights into age-related immune changes:
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Immunosenescence research: Studies have shown that intrinsic TGF-beta signaling contributes to age-dependent attrition of CD8+ T cell polyfunctionality
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Comparative analysis: The antibody allows comparison of TGF-beta RII expression between young and aged immune cells
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Functional correlation: Expression levels detected by the antibody can be correlated with functional immune parameters
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Mechanistic studies: The antibody helps elucidate the molecular mechanisms underlying age-related immune dysfunction
Research using this antibody has revealed that TGF-beta signaling changes with age contribute to altered immune function, particularly in CD8+ T cells, offering potential therapeutic targets for age-related immune decline .
