Phospho-FCGR2B (Y292) Antibody

What is FCGR2B and why is the Y292 phosphorylation site important?

FCGR2B (CD32B) functions as a low-affinity receptor for the Fc region of complexed or aggregated immunoglobulins gamma. It contains a cytoplasmic immunoreceptor tyrosine-based inhibitor motif (ITIM) that becomes phosphorylated at Y292, enabling binding to SH2 domain-containing phosphatases . This phosphorylation is critical because it initiates inhibitory signaling cascades that down-modulate cell activation previously triggered via antigen receptors on B-cells (BCR), T-cells (TCR), or other Fc receptors . The Y292 site serves as a molecular switch that controls the receptor's inhibitory function in immune regulation.

Which experimental models are suitable for studying phospho-FCGR2B (Y292)?

Multiple experimental models can be employed to study phospho-FCGR2B (Y292), including:

• Cell lines: K562 cells treated with PMA have been validated for western blot applications with phospho-Y292 antibodies .

• Primary human immune cells: B cells, monocytes, neutrophils, macrophages, basophils, eosinophils, and Langerhans cells all express FCGR2B .

• Mouse models: Both wild-type and transgenic mice expressing human FCGR2B can be valuable for in vivo studies .

• In vitro stimulation models: Multivalent Fc-containing molecules can induce FCGR2B phosphorylation in controlled systems .

When selecting an experimental model, researchers should consider species compatibility, as some phospho-FCGR2B antibodies demonstrate cross-reactivity across human, mouse, and rat systems, while others are species-specific .

How do I detect phosphorylated FCGR2B (Y292) in my samples?

Detection of phosphorylated FCGR2B (Y292) can be accomplished through several techniques:

Western Blot Protocol:

• Stimulate cells with appropriate activators (e.g., PMA for K562 cells or pervanadate for MCF-7 cells)

• Lyse cells in buffer containing phosphatase inhibitors

• Resolve proteins on 4-12% SDS-PAGE gels

• Transfer to nitrocellulose membranes

• Block with appropriate blocking buffer

• Incubate with anti-phospho-Y292 FCGR2B antibody at recommended dilutions (typically 1:300-1:1000)

• Detect using appropriate secondary antibodies and imaging systems

• Include total FCGR2B and loading controls (β-actin or total Akt)

Additional Methods:

• Flow cytometry: Useful for cellular detection using dilutions of approximately 1:50-1:200

• Immunofluorescence: Enables visualization of subcellular localization

• ELISA: Allows quantitative measurement in solution

What are common challenges in detecting phospho-FCGR2B (Y292) and how can they be addressed?

Several challenges may arise when detecting phospho-FCGR2B (Y292):

• Rapid dephosphorylation: Phosphorylation events are often transient. Use phosphatase inhibitors (such as sodium orthovanadate, sodium fluoride, and β-glycerophosphate) in all buffers immediately after cell stimulation .

• Low signal-to-noise ratio: Optimize antibody concentration, incubation time, and washing steps. Consider using enhanced chemiluminescent substrates for western blot applications.

• Cross-reactivity concerns: Validate specificity using appropriate controls, including:

  • Unstimulated cells (negative control)

  • Samples treated with phosphatase

  • Competitive blocking with immunizing peptide

• Variability across species: Ensure the antibody is validated for your species of interest. While some antibodies recognize human, mouse, and rat phospho-FCGR2B, others may be species-specific .

• Inconsistent stimulation: Standardize stimulation protocols with positive controls like pervanadate treatment, which broadly inhibits tyrosine phosphatases .

How does Fc sialylation affect FCGR2B phosphorylation and what methods can detect this relationship?

Recent research has revealed that Fc sialylation significantly impacts FCGR2B phosphorylation, particularly when Fc domains are presented in a multivalent rather than monovalent fashion . This relationship appears crucial for the anti-inflammatory activity of various therapeutic antibodies.

Methodological approach to study this relationship:

• Preparation of differentially sialylated Fc-containing molecules:

  • Use enzymatic glycoengineering to create antibodies with controlled sialylation levels

  • Generate recombinant multivalent Fc products (e.g., Fc3Y, Fc5X, Fc5Y) as described by Ortiz (2016)

  • Label constructs with fluorescent tags (e.g., VivoTag 645) for tracking

• Multivalent display systems:

  • Create immune complexes by mixing anti-TNFα antibodies with soluble trimeric TNFα in a 3:1 molar ratio

  • Incubate mixtures at 37°C for 24-48 hours to form stable complexes

  • Validate complex formation using Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS)

• Phosphorylation detection:

  • Stimulate human cellular systems (e.g., B cells at 1×10^6 cells/well) with the sialylated complexes for 30 minutes at 37°C

  • Analyze phospho-Y292 FCGR2B using western blot with densitometry measurements

Research has demonstrated that multivalent Fcs lead to robust FCGR2B phosphorylation in a sialic acid-dependent manner, providing new insights into potential anti-inflammatory mechanisms of therapeutic antibodies .

What are the best practices for quantifying phospho-FCGR2B (Y292) signals in western blot experiments?

Accurate quantification of phospho-FCGR2B (Y292) signals requires rigorous experimental design and analysis:

• Standardization and normalization:

  • Always include total FCGR2B detection on the same or parallel blots

  • Normalize phospho-signal to total protein signal

  • Include housekeeping controls such as β-actin or total Akt

  • Perform technical replicates (minimum of three)

• Densitometry analysis:

  • Use infrared imaging systems (e.g., Odyssey CLx) for wider dynamic range and greater sensitivity

  • Ensure signal falls within the linear range of detection

  • Subtract background using appropriate controls

  • Report data as fold-change relative to unstimulated conditions

• Statistical analysis:

  • Use appropriate statistical tests (e.g., t-test for two conditions, ANOVA for multiple conditions)

  • Report standard deviation or standard error

  • Consider power analysis to determine adequate sample sizes

• Controls to include:

  • Unstimulated cells (negative control)

  • Positive control (e.g., pervanadate-treated cells)

  • Isotype control antibody

  • Peptide competition control

How can I study the relationship between FCGR2B phosphorylation and downstream signaling pathways?

Investigating the relationship between FCGR2B phosphorylation and downstream signaling requires sophisticated experimental approaches:

• Temporal signaling dynamics:

  • Perform time-course experiments (0-60 minutes) following stimulation

  • Monitor sequential phosphorylation events of downstream mediators

  • Use phospho-specific antibodies targeting SH2-containing phosphatases (SHIP, SHP-1, SHP-2)

• Inhibitor studies:

  • Apply selective inhibitors of tyrosine kinases (e.g., SRC family inhibitors)

  • Use phosphatase inhibitors to block signal termination

  • Employ genetic approaches (siRNA, CRISPR) to validate key signaling components

• Protein-protein interaction analysis:

  • Perform co-immunoprecipitation of FCGR2B with SH2-containing phosphatases

  • Use proximity ligation assays to visualize interactions in situ

  • Apply FRET-based approaches to monitor real-time interactions

• Functional readouts:

  • Measure calcium flux inhibition

  • Analyze B cell receptor signaling modulation

  • Assess effects on antibody production or phagocytosis

This integrated approach allows for comprehensive mapping of how phosphorylation at Y292 initiates and regulates inhibitory signaling cascades.

What methodological approaches can resolve discrepancies in phospho-FCGR2B detection across different experimental systems?

Researchers occasionally encounter contradictory results when studying phospho-FCGR2B across different experimental systems. Several methodological approaches can help resolve these discrepancies:

• Antibody validation panel:

  • Test multiple phospho-specific antibodies from different vendors

  • Compare monoclonal vs. polyclonal antibodies

  • Validate specificity using phosphatase treatment and blocking peptides

  • Consider using antibodies raised against different epitopes surrounding Y292

• Cross-platform validation:

  • Confirm key findings using complementary techniques (e.g., mass spectrometry, phospho-flow cytometry)

  • Employ phosphoproteomics to identify all phosphorylation sites simultaneously

  • Use CRISPR-engineered Y292F mutants as negative controls

• Standardization of stimulation conditions:

  • Create detailed protocols specifying cell density, stimulation time, temperature

  • Control for receptor expression levels across systems

  • Consider the impact of multivalent vs. monovalent stimulation

• Species-specific considerations:

  • Recognize that human and murine FCGR2B may exhibit subtle differences in phosphorylation dynamics

  • Use species-matched reagents throughout experiments

  • Consider generating species-specific antibodies for cross-species studies

How can phospho-FCGR2B (Y292) antibodies be utilized in studying autoimmune conditions?

FCGR2B plays a critical role in preventing autoimmunity, with genetic variants associated with systemic lupus erythematosus (SLE) . Phospho-FCGR2B (Y292) antibodies provide valuable tools for investigating autoimmune mechanisms:

• Patient sample analysis:

  • Compare phospho-FCGR2B levels in B cells from healthy donors versus SLE patients

  • Correlate phosphorylation status with disease activity markers

  • Assess the impact of treatments on FCGR2B phosphorylation

• Functional studies:

  • Investigate how autoimmune-associated FCGR2B variants affect Y292 phosphorylation

  • Examine phosphorylation-dependent inhibitory function in B cells from patients

  • Assess the relationship between phospho-FCGR2B levels and autoantibody production

• Animal models:

  • Use phospho-Y292 antibodies in mouse models of SLE and other autoimmune conditions

  • Track phosphorylation changes during disease progression

  • Test therapeutic strategies targeting the FCGR2B phosphorylation pathway

• Biomarker development:

  • Evaluate phospho-FCGR2B (Y292) as a potential biomarker for treatment response

  • Develop standardized assays for clinical sample testing

  • Correlate phosphorylation levels with disease subtypes

What are the methodological considerations when studying phospho-FCGR2B (Y292) in cancer immunotherapy research?

FCGR2B has emerged as an important factor in antibody-mediated tumor control, particularly in the context of therapeutic monoclonal antibodies like rituximab . Key methodological considerations include:

• Tumor cell analysis:

  • Assess FCGR2B expression and phosphorylation status in different tumor types

  • Compare phospho-FCGR2B levels before and after antibody therapy

  • Investigate correlation between phosphorylation status and therapy resistance

• Dual targeting strategies:

  • Test combined use of anti-tumor antibodies with FCGR2B-blocking antibodies

  • Analyze phospho-Y292 as a biomarker for response to dual targeting

  • Develop phospho-specific inhibitors of FCGR2B signaling

• Adoptive transfer models:

  • Use models where human FCGR2B is expressed only on target cells

  • Monitor depletion of adoptively transferred B cells or tumor cells

  • Investigate depletion efficiency with clinically relevant anti-CD20 antibodies

• Pharmacodynamic assessment:

  • Monitor phospho-FCGR2B status as a pharmacodynamic biomarker

  • Correlate with antibody half-life and serum exposure

  • Assess impact on therapeutic efficacy in vivo

Proper implementation of these methodologies can provide insights into how FCGR2B phosphorylation affects cancer immunotherapy outcomes and potentially identify strategies to overcome resistance mechanisms.

How should phospho-FCGR2B (Y292) antibodies be optimally stored and handled for research applications?

Proper storage and handling of phospho-FCGR2B (Y292) antibodies are critical for maintaining specificity and sensitivity:

Additional handling recommendations:

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

• Avoid contamination by using sterile pipette tips

• Return to appropriate storage temperature immediately after use

• Monitor expiration dates and antibody performance over time

• Consider including positive controls with each experiment to verify antibody functionality

What validation experiments should be performed when working with a new phospho-FCGR2B (Y292) antibody?

Before implementing a new phospho-FCGR2B (Y292) antibody in critical experiments, comprehensive validation is essential:

• Specificity validation:

  • Stimulate cells known to express FCGR2B with appropriate activators (e.g., PMA, pervanadate)

  • Compare with unstimulated controls

  • Perform phosphatase treatment to confirm signal represents phosphorylation

  • Use peptide competition with the immunizing phosphopeptide

  • If possible, use FCGR2B knockout or Y292F mutant cells as negative controls

• Application-specific validation:

  • For western blot: Confirm single band at expected molecular weight (~34 kDa)

  • For immunoprecipitation: Verify enrichment of phosphorylated protein

  • For flow cytometry: Test fixation and permeabilization conditions

  • For immunofluorescence: Optimize fixation to preserve phospho-epitopes

• Technical optimization:

  • Test a range of antibody dilutions beyond manufacturer recommendations

  • Optimize blocking conditions to minimize background

  • Compare different detection systems for sensitivity

  • Determine limits of detection using serial dilutions of positive control lysates

• Cross-reactivity assessment:

  • Test reactivity with related phospho-tyrosine motifs

  • Confirm specificity across relevant species (human, mouse, rat)

  • Evaluate potential cross-reactivity with other FCGR family members

Thorough validation ensures reliable and reproducible results in subsequent experiments.

What are the most sensitive methods for detecting low levels of phospho-FCGR2B (Y292) in complex biological samples?

Detecting low abundance phospho-FCGR2B (Y292) in complex samples requires specialized approaches:

• Enhanced western blot techniques:

  • Use high-sensitivity chemiluminescent substrates

  • Apply signal amplification systems

  • Consider capillary western systems (e.g., Jess, ProteinSimple) for higher sensitivity

  • Implement sample enrichment by immunoprecipitation prior to western blotting

• Phospho-flow cytometry:

  • Optimize fixation and permeabilization protocols specifically for phospho-epitopes

  • Use fluorophores with high quantum yield

  • Implement signal amplification techniques (e.g., tyramide signal amplification)

  • Apply barcoding strategies for improved sample comparison

• Mass spectrometry approaches:

  • Enrich phosphopeptides using titanium dioxide or immobilized metal affinity chromatography

  • Apply targeted mass spectrometry (parallel reaction monitoring)

  • Use heavy isotope-labeled phosphopeptide standards for absolute quantification

  • Consider phospho-specific immunoaffinity enrichment prior to MS analysis

• Proximity ligation assay (PLA):

  • Detect interaction between phospho-Y292 and SH2-containing phosphatases

  • Provides single-molecule sensitivity with spatial resolution

  • Allows in situ detection in tissue sections

  • Minimizes background through dual antibody recognition requirement

These advanced methods can significantly improve detection limits while maintaining specificity for phospho-FCGR2B (Y292).

How can phospho-FCGR2B (Y292) antibodies be used to evaluate therapeutic antibody efficacy?

Phospho-FCGR2B (Y292) antibodies serve as valuable tools for evaluating therapeutic antibody mechanisms and efficacy:

• Mechanism of action studies:

  • Assess how therapeutic antibodies like rituximab affect FCGR2B phosphorylation

  • Investigate whether Fc sialylation in therapeutic antibodies influences FCGR2B phosphorylation

  • Determine if FCGR2B phosphorylation correlates with clinical response

• Combination therapy development:

  • Test how FCGR2B-blocking antibodies (e.g., 6G11, 6G, BI-1206) affect phospho-Y292 levels

  • Evaluate phosphorylation changes when combining multiple therapeutic antibodies

  • Develop biomarker assays based on phospho-FCGR2B status

• Fc engineering applications:

  • Compare how different Fc modifications impact FCGR2B phosphorylation

  • Assess the relationship between Fc sialylation and inhibitory signaling

  • Optimize therapeutic antibodies to modulate FCGR2B phosphorylation appropriately

• Predictive biomarker development:

  • Correlate baseline phospho-FCGR2B levels with treatment outcomes

  • Develop standardized assays for clinical implementation

  • Establish threshold values that predict therapeutic response

Understanding how therapeutic antibodies affect FCGR2B phosphorylation can guide the development of next-generation antibody therapeutics with improved efficacy.

What are the key methodological considerations when studying the impact of FCGR2B phosphorylation on B cell depletion therapies?

B cell depletion therapies, such as anti-CD20 antibodies, are significantly influenced by FCGR2B function. Key methodological considerations include:

• In vitro depletion assays:

  • Set up ADCC (antibody-dependent cellular cytotoxicity) assays with effector cells

  • Monitor FCGR2B phosphorylation status during depletion

  • Compare wild-type vs. Y292F mutant FCGR2B expression systems

  • Assess internalization of antibody-antigen complexes in relation to phospho-FCGR2B levels

• In vivo models:

  • Use transgenic mice expressing human FCGR2B

  • Perform adoptive transfer of target cells expressing different levels of FCGR2B

  • Monitor depletion efficiency in various tissues

  • Track phospho-FCGR2B status before and after antibody treatment

• Receptor distribution analysis:

  • Assess FCGR2B expression and phosphorylation across different B cell subsets

  • Examine the distribution between target cells and effector cells

  • Investigate how receptor density affects phosphorylation efficiency

  • Study the importance of "cis" interactions that may cause internalization of CD20:mAb complexes

• Antibody exposure dynamics:

  • Evaluate whether FCGR2B phosphorylation affects antibody half-life

  • Measure serum exposure in different experimental contexts

  • Correlate phosphorylation status with antibody clearance rates

These methodological approaches provide a comprehensive framework for understanding how FCGR2B phosphorylation impacts the efficacy of B cell depletion therapies.