RASGRP2 Antibody

What is RASGRP2 and what cellular functions does it regulate?

RASGRP2, also known as CalDAG-GEFI, CDC25L, and MCG7, belongs to the RASGRP family and functions primarily as a calcium- and diacylglycerol (DAG)-regulated guanine nucleotide exchange factor (GEF). It specifically activates Rap through the exchange of bound GDP for GTP and may also activate other GTPases such as RRAS, RRAS2, NRAS, and KRAS, but not HRAS .

RASGRP2 plays critical roles in:

• Aggregation of platelets through inside-out integrin activation

• Adhesion of T-lymphocytes and neutrophils

• The muscarinic acetylcholine receptor M1/CHRM1 signaling pathway

• Suppression of Bax activation-induced apoptosis via R-Ras-PI3K-Akt signaling

The protein has a calculated molecular weight of 69 kDa, though it may be observed at different weights in various experimental contexts (48-69 kDa) .

What are the recommended applications for RASGRP2 antibodies?

RASGRP2 antibodies have been validated for multiple experimental applications with specific recommended protocols:

It is strongly recommended that researchers titrate antibodies in their specific testing systems to optimize results, as antibody performance can be sample-dependent .

What cell and tissue types express RASGRP2?

RASGRP2 expression has been validated in multiple cell types, with particularly robust expression in:

• Platelets (critical for platelet aggregation and function)

• T-lymphocytes (important for adhesion functions)

• Neutrophils (regulates β1 and β2 integrin expression and function)

• Human umbilical vein endothelial cells (HUVECs)

For antibody validation, positive Western blot results have been detected in:

• Jurkat cells (T lymphocyte line)

• MOLT-4 cells (acute lymphoblastic leukemia)

• Raji cells (Burkitt's lymphoma)

Recent research has also identified important roles for RASGRP2 in lung adenocarcinoma (LUAD), where it functions as a potential immune-related biomarker .

How should RASGRP2 antibodies be stored and handled to maintain optimal activity?

For optimal RASGRP2 antibody stability and performance, the following storage and handling protocols are recommended:

• Store at -20°C in the buffer provided (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Antibodies remain stable for one year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage

• Some preparations (20μl sizes) may contain 0.1% BSA as a stabilizer

When working with the antibody:

• Avoid repeated freeze-thaw cycles

• Allow the antibody to equilibrate to room temperature before opening

• Centrifuge briefly before use to collect solution at the bottom of the vial

• Return to -20°C immediately after use

Antibody precipitation may indicate reduced stability or compromised function, as observed with some RASGRP2 mutants that showed increased precipitation during purification processes .

What are the most effective validation methods for RASGRP2 antibodies?

Thorough validation of RASGRP2 antibodies should include:

• Western Blot Validation:

  • Confirm detection at the expected molecular weight (calculated: 69 kDa)

  • Note that observed molecular weight may vary (48-69 kDa depending on antibody clone)

  • Include positive controls such as Jurkat, MOLT-4, or Raji cells

  • Include negative controls and/or knockout/knockdown samples if available

• Specificity Testing:

  • Test reactivity across species (human, mouse) if cross-reactivity is claimed

  • Validate using peptide blocking experiments with the immunogen

  • Ideally compare results with multiple RASGRP2 antibodies targeting different epitopes

• Functional Validation:

  • For flow cytometry, compare expression in known positive cells (e.g., Jurkat) versus negative controls

  • For CoIP experiments, validate interaction partners documented in literature

  • Consider validating with recombinant RASGRP2 protein as a positive control

Include parallel testing of related proteins (e.g., Rap1, Rasa3) to ensure specificity, as demonstrated in studies of RASGRP2 mutations .

How can I optimize Western blot protocols for RASGRP2 detection?

For optimal Western blot detection of RASGRP2, consider the following protocol adjustments:

• Sample Preparation:

  • For platelet samples: Careful isolation of platelets to avoid activation

  • For cell lines: Jurkat, MOLT-4, and Raji cells serve as positive controls

  • Include protease inhibitor cocktails in lysis buffers

  • Consider phosphatase inhibitors if examining phosphorylation states

• Gel Electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Load appropriate positive controls alongside experimental samples

  • Expected molecular weight: 69 kDa (calculated), though 48 kDa has been observed with some antibodies

• Antibody Incubation:

  • Primary antibody dilution: Start with 1:1000-1:6000 range

  • Incubate overnight at 4°C for optimal binding

  • Include proper blocking (5% non-fat milk or BSA in TBST)

  • Extensive washing steps (at least 3-5 times for 5-10 minutes each)

• Detection Optimization:

  • For low expression samples, consider enhanced chemiluminescence (ECL) substrates

  • Secondary antibody selection should match host species (typically anti-rabbit IgG)

  • Adjust exposure times based on signal intensity

If weak or nonspecific signals are observed, further optimization through antibody titration and adjustment of blocking conditions may be necessary.

How does RASGRP2 regulate apoptosis through the R-Ras-PI3K-Akt signaling pathway?

RASGRP2 plays a critical role in suppressing Bax activation-induced apoptosis through a mechanism independent of its well-known Rap1 activation function. Research using BAM7 and anisomycin (apoptosis inducers) has revealed:

• R-Ras Activation Mechanism:

  • RASGRP2 activates not only Rap1 but also R-Ras

  • Unlike Rap1-mediated effects, RASGRP2's anti-apoptotic function persists even when Rap1 is knocked down

  • This indicates a separate R-Ras-dependent pathway for apoptosis regulation

• Signaling Cascade:

  • RASGRP2 → R-Ras activation → PI3K stimulation → Akt phosphorylation

  • Phosphorylated Akt inhibits Bax translocation to mitochondria

  • This occurs through promoting hexokinase-2 (HK-2) translocation from cytoplasm to mitochondria

• Experimental Evidence:

  • RASGRP2-stable overexpression in immortalized HUVECs (R cells) showed significant protection against BAM7- and anisomycin-induced apoptosis compared to Mock cells

  • Importantly, this protection operated independently of the ROS production pathway, distinguishing it from TNF-α-induced apoptosis mechanisms

This pathway represents a novel function of RASGRP2 in cellular protection and suggests that RASGRP2 antibodies may be valuable tools for investigating mitochondrial-dependent apoptosis mechanisms in various disease contexts.

What is the significance of RASGRP2 mutations in platelet function disorders?

RASGRP2 mutations have been identified as important causes of inherited platelet function disorders characterized by bleeding diathesis. Key findings include:

• Clinical Presentation:

  • Patients with homozygous RASGRP2 mutations display lifelong bleeding tendencies

  • Symptoms include spontaneous bruising, petechiae, epistaxis, gingival and gastrointestinal bleeding

  • Interestingly, bleeding complications appear less severe in adults

• Molecular Mechanisms:

  • Mutations in RASGRP2 impair CalDAG-GEFI expression and/or function

  • This leads to defective αIIbβ3 integrin activation in platelets

  • Multiple mutation types have been identified, including:

    • p.Ser381Phe: Causes conformational changes affecting protein stability and nucleotide exchange activity

    • p.Arg113X: Results in markedly reduced CalDAG-GEFI expression

    • p.Gly248Trp: Positioned at the interface between CDC25 domain and Rap1

• Functional Consequences:

  • Markedly impaired platelet aggregation in response to low concentrations of agonists (particularly ADP and collagen)

  • Delayed aggregation even with high concentrations of most agonists

  • Alterations in β1 and β2 integrin expression/function in neutrophils

• Geographic Distribution:

  • The first comprehensive Indian study identified eight patients with RASGRP2 variants associated with platelet function defects

  • Previously reported in other populations, showing the global relevance of these mutations

RASGRP2 antibodies are valuable tools for characterizing these disorders, particularly for assessing protein expression levels in patient platelets through Western blotting and other techniques.

How is RASGRP2 implicated in cancer biology, particularly in lung adenocarcinoma?

Recent research has revealed important connections between RASGRP2 and cancer biology, with particular significance in lung adenocarcinoma (LUAD):

• Expression Patterns:

  • Decreased RASGRP2 expression is associated with worse clinical parameters and poorer prognosis in LUAD patients

  • RASGRP2 appears to function as a potential tumor suppressor in this context

• Immune Regulation:

  • RASGRP2 is involved in lymphocyte activation and leukocyte adhesion

  • It positively correlates with infiltration of most immune cells, immunoregulators, and chemokines

  • Expression levels of PDCD1, CTLA4, CD40LG, CCL14, CXCR5, and CCR7 are higher in high-RASGRP2 expression groups

• Molecular Mechanisms:

  • A FLI1-HSA-miR-1976-RASGRP2 transcriptional network has been identified

  • RASGRP2 inhibits cell proliferation in LUAD through regulation of mitochondrial-dependent apoptosis

  • This provides a mechanistic link to its anti-tumor properties

• Research Applications:

  • RASGRP2 antibodies are essential tools for studying these mechanisms

  • Particularly useful for:

    • Expression analysis in tumor samples

    • Correlation with immune cell infiltration markers

    • Mechanistic studies of mitochondrial-dependent apoptosis

These findings suggest RASGRP2 could serve as a potential immune-related biomarker in LUAD and potentially other cancers, making RASGRP2 antibodies valuable tools for cancer immunology research.

What are the critical considerations when using RASGRP2 antibodies for flow cytometry?

When using RASGRP2 antibodies for flow cytometry, researchers should consider several critical factors:

• Sample Preparation:

  • RASGRP2 requires intracellular staining protocols as it is not a cell surface protein

  • Validated in Jurkat cells at a concentration of 0.40 μg per 10^6 cells in a 100 μl suspension

  • Proper fixation and permeabilization are essential for antibody access to intracellular targets

• Antibody Selection:

  • Choose antibodies specifically validated for flow cytometry applications

  • Consider clone specificity and background signal characteristics

  • For dual staining experiments, select antibodies with compatible fluorophores and minimal spectral overlap

• Protocol Optimization:

  • Titrate antibody concentrations to determine optimal signal-to-noise ratio

  • Include proper blocking steps to minimize non-specific binding

  • Use appropriate isotype controls matched to the primary antibody

  • Set up compensation properly when using multiple fluorochromes

• Data Interpretation:

  • RASGRP2 should show primarily intracellular localization

  • Compare expression patterns with known positive controls (e.g., Jurkat cells)

  • Consider analyzing correlation with activation status in immune cells or platelets

  • When studying mutations, compare with wild-type expression patterns as reference

• Special Considerations:

  • For platelet studies, careful activation control is critical as activation state affects RASGRP2 localization

  • For cancer studies, correlation with other markers may provide functional insights

  • When examining neutrophils, consider concurrent analysis of integrin activation markers

These technical considerations will help ensure reliable and reproducible results when examining RASGRP2 expression and function using flow cytometry.

How can I address common issues when working with RASGRP2 antibodies?

Researchers may encounter several challenges when working with RASGRP2 antibodies. Here are solutions to common problems:

If protein precipitation occurs during purification or experiments (as observed with some RASGRP2 mutants), this may indicate compromised protein stability and require adjustment of buffer conditions or experimental approach .

What are the best positive and negative controls for RASGRP2 antibody validation?

Selecting appropriate controls is critical for validating RASGRP2 antibodies across different applications:

Positive Controls:

• Cell Lines:

  • Jurkat cells (T lymphocyte line) - Confirmed for WB and FC applications

  • MOLT-4 cells (acute lymphoblastic leukemia)

  • Raji cells (Burkitt's lymphoma)

• Tissue Samples:

  • Platelets - High RASGRP2 expression

  • T-lymphocytes - Known expression

  • Neutrophils - Validated expression

• Recombinant Proteins:

  • Purified wild-type RASGRP2 protein

  • Tagged RASGRP2 expression constructs

Negative Controls:

• Methodological Controls:

  • Secondary antibody only

  • Isotype control antibodies

  • Pre-immune serum (for polyclonal antibodies)

• Genetic Controls:

  • RASGRP2 knockout or knockdown cells

  • Cells expressing mutant RASGRP2 with reduced stability (e.g., p.Ser381Phe mutant)

• Blocking Controls:

  • Pre-incubation of antibody with immunizing peptide

  • Competition assays with unlabeled antibody

Validation Approaches:

• Compare antibody performance across multiple sample types

• Verify specificity across species if cross-reactivity is claimed

• Confirm detection at expected molecular weight (calculated: 69 kDa, though 48 kDa has been observed with some antibodies)

• Test across multiple applications to ensure consistent results

These controls help ensure antibody specificity and reliability across experimental applications.

How can RASGRP2 antibodies be used effectively in co-immunoprecipitation experiments?

Co-immunoprecipitation (CoIP) with RASGRP2 antibodies can provide valuable insights into protein-protein interactions. Here's a methodological approach for effective CoIP experiments:

• Antibody Selection:

  • Choose antibodies specifically validated for CoIP applications

  • Consider using antibodies targeting different RASGRP2 epitopes for confirmation

  • For challenging interactions, consider tagged constructs as alternatives

• Sample Preparation:

  • Cell lysis conditions are critical:

    • Use gentle, non-ionic detergents (0.5-1% NP-40 or Triton X-100)

    • Include protease and phosphatase inhibitors

    • Perform lysis at 4°C to preserve protein-protein interactions

  • Pre-clear lysates with appropriate control beads/antibodies

• Immunoprecipitation Protocol:

  • Antibody binding:

    • Incubate antibody with lysate (typically 2-5 μg per 500 μg protein)

    • Overnight incubation at 4°C with gentle rotation

  • Capture with protein A/G beads:

    • 1-2 hours incubation with beads

    • Gentle washing (3-5 times) with cold buffer

  • Elution:

    • Gentle elution to preserve interactions

    • SDS-PAGE sample buffer for Western blot analysis

• Controls and Validation:

  • Input controls (5-10% of starting material)

  • IgG control (same species as RASGRP2 antibody)

  • Reverse CoIP where possible

  • Validation of key interactions:

    • RASGRP2-Rap1 interaction

    • RASGRP2-R-Ras interaction

    • Other GTPases (RRAS2, NRAS, KRAS) as relevant

• Analytical Considerations:

  • Western blot detection of co-precipitated proteins

  • Consider mass spectrometry for unbiased interaction screening

  • Functional validation of identified interactions

This approach will help identify and validate RASGRP2 interaction partners, contributing to a better understanding of its role in various signaling pathways, particularly in platelet function and apoptosis regulation.

How is RASGRP2 being studied in the context of targeted therapies?

RASGRP2 is emerging as a potential therapeutic target with several promising research directions:

• Platelet Disorders and Bleeding Risk:

  • RASGRP2 mutations are associated with bleeding disorders characterized by impaired platelet function

  • Understanding RASGRP2 function could lead to:

    • Improved diagnostics for inherited platelet disorders

    • Novel therapeutic approaches to modulate platelet activation

    • Potential biomarkers for bleeding risk assessment in clinical settings

• Cancer Immunotherapy Connections:

  • RASGRP2 functions as a potential immune-related biomarker in lung adenocarcinoma (LUAD)

  • Research indicates RASGRP2 is positively correlated with:

    • Infiltration of immune cells

    • Expression of immunoregulators and chemokines

    • Levels of important immune checkpoint molecules (PDCD1, CTLA4)

  • These findings suggest RASGRP2 could be:

    • A predictive biomarker for immunotherapy response

    • A target for enhancing immune infiltration in "cold" tumors

• Anti-Apoptotic Mechanisms:

  • RASGRP2 inhibits cell proliferation in LUAD through regulation of mitochondrial-dependent apoptosis

  • The R-Ras-PI3K-Akt signaling pathway regulated by RASGRP2 represents a potential therapeutic target

  • Modulating RASGRP2-dependent hexokinase-2 (HK-2) translocation to mitochondria could offer new approaches to cancer treatment

RASGRP2 antibodies are essential tools for advancing these research directions, particularly for assessing protein expression, localization, and interaction with potential therapeutic targets.

What are the latest findings regarding RASGRP2 mutations and their clinical implications?

Recent research has expanded our understanding of RASGRP2 mutations and their clinical significance:

These findings highlight the importance of continued research into RASGRP2 mutations for both diagnostic and therapeutic applications.

How do different RASGRP2 antibody clones compare in research applications?

Different RASGRP2 antibody clones exhibit varying characteristics that can significantly impact research applications:

Comparative Analysis:

• Application Suitability:

  • For Western blotting: 30189-1-AP and 19745-1-AP are well-validated

  • For intracellular flow cytometry: 30189-1-AP is specifically validated

  • For CoIP applications: 30189-1-AP has published validation

• Species Considerations:

  • For human-only studies: Any of the listed antibodies

  • For cross-species studies (human/mouse): 19745-1-AP offers broader reactivity

• Epitope Recognition:

  • C-terminal targeting (19745-1-AP) may be advantageous for detecting truncation mutations

  • Different epitope recognition can affect detection of specific post-translational modifications

• Molecular Weight Variations:

  • Notable differences in observed molecular weight (48-69 kDa)

  • May reflect detection of different isoforms or post-translational modifications

  • Important consideration when interpreting experimental results

When selecting an antibody, researchers should consider these differences and choose the most appropriate clone based on their specific experimental requirements, target species, and detection method.

What are the recommended protocols for immunohistochemical detection of RASGRP2?

For effective immunohistochemical (IHC) detection of RASGRP2 in tissue samples, the following protocol is recommended:

• Tissue Preparation:

  • Fixation: 10% neutral buffered formalin (24-48 hours)

  • Processing: Standard paraffin embedding

  • Sectioning: 4-5 μm thick sections

  • Mounting: Positively charged slides

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER):

    • Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

    • Pressure cooker: 125°C for 3 minutes or

    • Microwave: 95-98°C for 15-20 minutes

  • Allow sections to cool to room temperature (20 minutes)

• Blocking and Antibody Incubation:

  • Peroxidase blocking: 3% H₂O₂ (10 minutes)

  • Protein blocking: 5% normal serum (1 hour)

  • Primary antibody:

    • Dilution: Start with manufacturer recommendations; titrate as needed

    • Incubation: Overnight at 4°C or 1-2 hours at room temperature

  • Washing: PBS-Tween (3 × 5 minutes)

  • Secondary antibody: HRP-conjugated (30-60 minutes)

  • Washing: PBS-Tween (3 × 5 minutes)

• Detection and Counterstaining:

  • DAB chromogen: Apply until signal develops (2-10 minutes)

  • Counterstain: Hematoxylin (30-60 seconds)

  • Dehydration: Ascending ethanol series

  • Clearing: Xylene

  • Mounting: Permanent mounting medium

• Controls and Validation:

  • Positive tissue controls: Lymphoid tissues, platelets

  • Negative controls: Primary antibody omission

  • Isotype controls: Matching antibody isotype

• Interpretation Considerations:

  • RASGRP2 typically shows cytoplasmic staining

  • Expression may be particularly relevant in:

    • Platelet-rich regions

    • Lymphoid tissues

    • Lung adenocarcinoma samples (where it may serve as an immune-related biomarker)

This protocol can be further optimized based on specific tissue types and research objectives. For lung adenocarcinoma studies, correlation with immune cell markers may provide valuable insights into the relationship between RASGRP2 expression and immune infiltration.

How can I design experiments to study RASGRP2's role in apoptotic pathways?

To investigate RASGRP2's role in apoptotic pathways, particularly its anti-apoptotic function via R-Ras-PI3K-Akt signaling, consider the following experimental design:

• Expression Modulation Studies:

  • Overexpression System:

    • Generate stable cell lines overexpressing RASGRP2 (follow model in )

    • Include vector control (Mock) cells as comparison

    • Consider creating mutant variants (e.g., those affecting R-Ras binding)

  • Knockdown/Knockout Approaches:

    • siRNA/shRNA targeting RASGRP2

    • CRISPR-Cas9 mediated knockout

    • Validate knockdown/knockout efficiency by Western blot with RASGRP2 antibodies

• Apoptosis Induction and Assessment:

  • Inducers:

    • BAM7 (direct Bax activator)

    • Anisomycin (protein synthesis inhibitor that induces apoptosis)

    • Compare with ROS-dependent inducers (e.g., TNF-α) to differentiate pathways

  • Measurement Methods:

    • Flow cytometry for Annexin V/PI staining

    • Caspase activity assays (Caspase 3/7)

    • TUNEL assay for DNA fragmentation

    • Western blot for cleaved PARP and cleaved caspases

• Pathway Analysis:

  • R-Ras Activation:

    • GTP-bound R-Ras pull-down assays

    • Compare with Rap1 activation to distinguish pathways

    • Use Rap1 knockdown to isolate R-Ras-dependent effects

  • PI3K-Akt Signaling:

    • Western blot for phosphorylated Akt

    • PI3K inhibitors (e.g., LY294002) to block pathway

    • Akt inhibitors to confirm downstream effects

• Mitochondrial Function Assessment:

  • Bax Translocation:

    • Subcellular fractionation and Western blot

    • Immunofluorescence microscopy for Bax localization

  • Hexokinase-2 (HK-2) Translocation:

    • Mitochondrial fractionation

    • Co-immunoprecipitation with mitochondrial markers

    • Immunofluorescence for co-localization

  • Mitochondrial Membrane Potential:

    • JC-1 or TMRM fluorescent dyes

    • Live-cell imaging of mitochondrial dynamics

• Validation in Disease Models:

  • Cancer Models:

    • Lung adenocarcinoma cell lines (connecting to findings in )

    • Correlation with patient survival data

  • Platelet Function:

    • Studies using platelets from patients with RASGRP2 mutations

    • Assessment of apoptotic susceptibility and mitochondrial function

These experimental approaches will help elucidate RASGRP2's role in apoptotic regulation and potentially identify new therapeutic targets for diseases involving dysregulated apoptosis.

What techniques can be used to study RASGRP2 in relation to immune cell infiltration in cancer?

To investigate RASGRP2's role in immune cell infiltration in cancer, particularly in lung adenocarcinoma where it serves as a potential immune-related biomarker, researchers can employ the following methodological approaches:

• Expression Analysis in Clinical Samples:

  • Immunohistochemistry (IHC):

    • Multi-staining approaches combining RASGRP2 with immune cell markers

    • Quantitative image analysis of staining patterns and co-localization

    • Correlation with patient outcomes and treatment responses

  • Transcriptomic Analysis:

    • RNA-seq of tumor samples with varying RASGRP2 expression

    • Correlation with immune gene signatures

    • Construction of transcriptional regulatory networks (e.g., FLI1-HSA-miR-1976-RASGRP2 network)

• Computational Approaches:

  • ESTIMATE Algorithm:

    • Analyze correlation between RASGRP2 and immune infiltration

    • Generate immune scores for tumor samples based on expression data

  • Single-Cell Analysis:

    • Single-cell RNA sequencing of tumor-infiltrating immune cells

    • Compare RASGRP2 expression across different immune populations

    • Identify cell-specific roles and regulatory mechanisms

• Functional Validation:

  • Co-culture Systems:

    • Establish co-cultures of cancer cells with immune components

    • Modulate RASGRP2 expression to assess effects on immune cell recruitment

    • Measure changes in cytokine/chemokine production

  • Migration and Adhesion Assays:

    • Transwell migration assays with conditioned media

    • Direct adhesion assays between immune and cancer cells

    • Integrin activation studies (connecting to RASGRP2's role in integrin regulation)

• Molecular Mechanisms:

  • Immune Checkpoint Expression:

    • Assess correlation between RASGRP2 and immune checkpoints (PDCD1, CTLA4)

    • Flow cytometry and Western blot validation of protein expression

    • Functional assays of T-cell activation with RASGRP2 modulation

  • Chemokine Regulation:

    • Analyze expression of chemokines (CCL14, CXCR5, CCR7) in relation to RASGRP2

    • Chemokine receptor signaling studies

    • Chemotaxis assays with conditioned media

• In Vivo Models:

  • Syngeneic Mouse Models:

    • Establish tumors with varying RASGRP2 expression levels

    • Analyze immune infiltration by flow cytometry and IHC

    • Test combination with immunotherapy approaches

  • Patient-Derived Xenografts:

    • Humanized mouse models to study human immune interactions

    • Correlation of RASGRP2 expression with infiltration patterns

    • Therapeutic intervention studies

These methodological approaches provide a comprehensive framework for understanding how RASGRP2 influences immune cell infiltration in cancer, potentially identifying new strategies for enhancing immunotherapy efficacy in patients with varying RASGRP2 expression levels.

What are the key considerations when selecting a RASGRP2 antibody for specific research applications?

When selecting a RASGRP2 antibody for research, consider these essential factors:

• Application Compatibility:

  • Western Blot: Most antibodies are validated; recommended dilutions range from 1:1000-1:6000

  • Flow Cytometry: Requires antibodies specifically validated for intracellular staining

  • Co-IP: Select antibodies validated for maintaining protein-protein interactions

  • IHC/IF: Consider epitope accessibility in fixed tissues

• Species Reactivity:

  • Human-only studies: Multiple options available

  • Mouse studies: Select antibodies with validated cross-reactivity (e.g., 19745-1-AP)

  • Cross-species comparisons: Ensure consistent epitope recognition across species

• Antibody Format:

  • Monoclonal (e.g., 1E5, 3D8): Higher specificity, consistent lot-to-lot performance

  • Polyclonal (e.g., 30189-1-AP, 19745-1-AP): May recognize multiple epitopes, potentially higher sensitivity

• Epitope Consideration:

  • C-terminal targeting antibodies may better detect truncation mutants

  • Consider epitope accessibility in your experimental system

  • For mutation studies, ensure the epitope is not affected by the mutation of interest

• Validation Status:

  • Published literature citations provide confidence in performance

  • Validated positive controls include Jurkat, MOLT-4, and Raji cells

  • Consider antibodies with validation in your specific experimental system

• Technical Specifications:

  • Storage requirements (typically -20°C)

  • Buffer composition (PBS with 0.02% sodium azide and 50% glycerol)

  • Concentration and formulation

  • Expected molecular weight (varies from 48-69 kDa depending on antibody)