PLCG2 Antibody

What is PLCG2 and why is it important in immunological research?

PLCG2 (Phospholipase C gamma 2) is an enzyme that plays a critical role in immune cell signaling pathways. It catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate second messengers inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG) . These messengers regulate cellular processes including calcium mobilization and protein kinase C activation, which are essential for immune cell function .

PLCG2 is particularly important in:

• B cell receptor (BCR) signaling and development

• Natural killer (NK) cell function

• Myeloid cell responses including monocytes, macrophages, and dendritic cells

• Inflammatory response regulation

Mutations in PLCG2 are associated with immune dysregulation syndromes, making it a significant target for immunological and autoinflammatory disease research .

Which validation methods should be used to confirm PLCG2 antibody specificity?

To confirm PLCG2 antibody specificity, researchers should employ multiple validation approaches:

• Knockout validation: Compare antibody reactivity between PLCG2 wild-type and knockout cell lines to verify specific binding

• Overexpression systems: Test antibody performance in cells transfected with PLCG2 expression vectors (e.g., EGFP-PLCG2 plasmid)

• Western blot analysis: Confirm single band at expected molecular weight (approximately 148 kDa)

• Immunoprecipitation followed by mass spectrometry: Verify pulled-down protein identity

• Epitope mapping: Ensure antibody recognizes the intended region of PLCG2 protein

Multiple antibody clones recognizing different epitopes should be tested when possible to increase confidence in results, particularly for phospho-specific antibodies targeting key residues like Y753, Y759, Y1197, and Y1217 .

What are the recommended applications for PLCG2 antibodies in immune cell research?

Based on the literature, PLCG2 antibodies are valuable tools for multiple applications in immune cell research:

Most validated in: Daudi cells, Raji cells, Ramos cells, and primary human lymphocytes .

How can PLCG2 antibodies be used to study downstream signaling pathways in immune cells?

PLCG2 antibodies can be used to map complex signaling networks through multiple methodologies:

• Phosphorylation status analysis:

  • Use phospho-specific antibodies targeting Y753, Y759, Y1197, and Y1217 to monitor activation

  • Combine with flow cytometry to quantify phosphorylation in heterogeneous cell populations

  • Western blot time-course experiments to determine kinetics of phosphorylation following receptor stimulation

• Signalosome complex analysis:

  • Immunoprecipitation with PLCG2 antibodies to capture interacting proteins

  • Mass spectrometry to identify novel binding partners

  • Co-immunoprecipitation to confirm association with known partners (Syk, Btk, CD19)

  • Proximity ligation assays to visualize protein-protein interactions in situ

• Functional readouts:

  • Calcium flux assays paired with PLCγ2 detection to correlate enzyme activation with calcium release

  • ERK phosphorylation assessment as a downstream readout

  • NF-κB and NFAT pathway activation monitoring through reporter assays

• Single-cell analysis:

  • Couple with scRNA-seq to correlate protein expression with transcriptional profiles

  • Mass cytometry to simultaneously measure multiple pathway components

These approaches have been successfully used to characterize various gain-of-function and loss-of-function PLCG2 mutations and their effects on immune signaling .

What protocols are recommended for detecting both wild-type and mutant PLCG2 proteins in patient samples?

For clinical research involving PLCG2 variants, the following protocols have been validated:

Protocol for peripheral blood mononuclear cells (PBMCs):

• Isolate PBMCs using density gradient centrifugation

• Perform BCR stimulation (anti-IgM for 5 minutes)

• Fix cells with paraformaldehyde (2-4%)

• Permeabilize with methanol for phospho-epitope exposure

• Stain with anti-PLCG2 and phospho-PLCG2 antibodies

• Analyze by flow cytometry or imaging cytometry

For genetic variant functional assessment:

• Generate mutant PLCG2 constructs using site-directed mutagenesis

• Express constructs in PLCG2-deficient cell lines (e.g., DT-40 B cells)

• Measure functional readouts:

  • B-cell receptor-induced calcium flux by flow cytometry

  • ERK phosphorylation by Western blot or flow cytometry

  • IP3 production using enzyme-linked immunosorbent assay

For patient cell immunoblotting:

• Lyse cells in RIPA buffer with phosphatase inhibitors

• Separate proteins on 4-20% SDS-PAGE gel

• Transfer to PVDF membrane

• Block and probe with anti-PLCG2 antibodies

• Use phospho-specific antibodies to detect activation status

These methods have successfully differentiated between gain-of-function (GOF) and loss-of-function (LOF) PLCG2 variants in patients with immune dysregulation .

How do phospho-specific PLCG2 antibodies differ in application from total PLCG2 antibodies?

For functional studies of PLCG2 mutations, a combination of both phospho-specific and total PLCG2 antibodies provides the most complete characterization of signaling abnormalities .

How can PLCG2 antibodies help differentiate between PLAID and APLAID syndromes in research?

PLCG2 antibodies are crucial tools for investigating the molecular mechanisms underlying PLAID and APLAID syndromes. These approaches help differentiate between these related but distinct conditions:

Functional characterization strategies:

• Domain-specific antibodies: Antibodies targeting the cSH2 domain can detect structural abnormalities in PLAID (typically affected by genomic deletions), while antibodies against catalytic domains help characterize APLAID (typically affected by point mutations)

• Temperature-dependent activation analysis:

  • For PLAID research: Monitor PLCG2 activation at reduced temperatures (4-20°C)

  • For APLAID research: Assess constitutive activation at physiological temperature

  • Use phospho-specific antibodies to compare activation patterns

• Downstream signaling profiling:

  • PLAID: Characterized by selective calcium signaling defects

  • APLAID: Shows enhanced signaling through multiple pathways (MAPK, NF-κB, NFAT)

  • Use antibody panels to monitor these pathways simultaneously

• Cell-type specific effects:

  • Compare PLCG2 activation in B cells versus NK cells and myeloid cells

  • PLAID typically shows B cell defects with preserved myeloid signaling

  • APLAID demonstrates broader signaling dysregulation

This multimodal antibody-based approach helps classify PLCG2 variants along the phenotypic spectrum from PLAID to APLAID, which is critical for understanding genotype-phenotype correlations in these disorders .

What are the challenges in using PLCG2 antibodies to study heterozygous loss-of-function variants?

Studying heterozygous PLCG2 loss-of-function (LOF) variants presents several technical challenges that require specific methodological solutions:

• Distinguishing mutant from wild-type protein:

  • Challenge: Both proteins are present in patient cells at similar molecular weights

  • Solution: Develop allele-specific antibodies or use epitope tagging in model systems

• Measuring partial functional defects:

  • Challenge: Heterozygous variants often show subtle signaling abnormalities

  • Solution: Use highly quantitative readouts such as flow cytometry or phospho-flow with precise gating strategies

• Cell type-specific effects:

  • Challenge: LOF variants may affect NK cells more than B cells

  • Solution: Compare antibody detection across multiple immune cell populations using multiparameter flow cytometry

• Dose-dependent signaling:

  • Challenge: Loss of one functional allele may be compensated by the wild-type allele

  • Solution: Titrate stimulation conditions to reveal signaling defects at submaximal stimulation

• Background genetic factors:

  • Challenge: Other genetic variants may influence PLCG2 phenotype

  • Solution: Use isogenic cell lines with CRISPR-engineered mutations for controlled studies

Research has shown that heterozygous PLCG2 LOF variants are associated with a novel form of immune dysregulation including humoral immune deficiency, autoinflammation, herpesvirus susceptibility, and NK cell dysfunction, highlighting the importance of developing robust methods to study these mutations .

How do researchers validate novel PLCG2 variants with functional assays using antibodies?

Validation of novel PLCG2 variants requires a systematic approach combining genetic analysis, molecular modeling, and functional studies with antibodies:

Complete validation protocol:

• Initial screening:

  • Bioinformatic analysis of variant (conservation, CADD score, SiPhy)

  • Structural modeling of mutation's potential impact on protein domains

  • Comparison with known pathogenic variants

• Expression system validation:

  • Generate mutant PLCG2 constructs using site-directed mutagenesis

  • Express in PLCG2-deficient cell lines (e.g., PLCG2-knockout DT-40 B cells)

  • Use anti-PLCG2 antibodies to confirm equivalent expression levels by Western blot

• Functional readouts:

  • Calcium flux assays following BCR stimulation

  • Phospho-ERK detection by flow cytometry

  • IP3 production measurement

  • NFAT and NF-κB reporter assays (for suspected gain-of-function variants)

• Protein-protein interaction studies:

  • Immunoprecipitation with anti-PLCG2 antibodies

  • Assess binding to upstream regulators (Syk, Btk, BLNK)

  • Evaluate autoinhibitory domain interactions in gain-of-function variants

• Patient cell validation:

  • Compare antibody staining patterns in patient cells versus controls

  • Measure multiple signaling nodes to detect pathway dysregulation

  • Perform cell-type specific analyses (B cells, NK cells, myeloid cells)

This approach has successfully classified variants as gain-of-function, loss-of-function, or benign, correlating with specific clinical phenotypes ranging from autoinflammation to antibody deficiency .

How should experimental controls be designed when studying PLCG2 variants with antibodies?

Robust experimental design for PLCG2 variant studies requires careful consideration of controls:

Essential controls for PLCG2 antibody experiments:

• Genetic controls:

  • PLCG2 knockout cells (negative control)

  • Wild-type PLCG2 expression (baseline control)

  • Known pathogenic variants (positive controls)

    • Gain-of-function: S707Y, L848P, M1141K for APLAID-like phenotypes

    • Loss-of-function: heterozygous LOF variants from validated cases

• Stimulation controls:

  • Unstimulated baseline

  • Dose titration of BCR stimulation (anti-IgM)

  • Time-course sampling to capture signaling kinetics

  • Pharmacological controls (e.g., PLC inhibitors like U73122)

• Antibody controls:

  • Isotype-matched control antibodies

  • Secondary antibody-only controls

  • Phospho-specific antibody validation with phosphatase treatment

  • Multiple antibody clones targeting different epitopes

• Cell type controls:

  • Non-B cells for B-cell specific phenotypes

  • Cell lines with varying PLCG2 expression levels

  • Primary cells from healthy donors spanning age ranges

• Experimental system validation:

  • Reproducibility across multiple biological replicates

  • Validation in both overexpression systems and primary cells

  • Cross-validation with functional readouts (calcium flux, pERK)

This comprehensive control strategy ensures reliable interpretation of PLCG2 antibody-based experiments, particularly when studying novel variants of uncertain significance .

What troubleshooting steps should be taken when PLCG2 antibodies show inconsistent results?

When facing inconsistent results with PLCG2 antibodies, follow this systematic troubleshooting approach:

Antibody-specific issues:

• Verify antibody quality:

  • Check antibody validation data using knockout controls

  • Test multiple antibody lots

  • Validate with another independent antibody targeting a different epitope

• Optimize antibody conditions:

  • Titrate antibody concentration

  • Modify incubation time and temperature

  • Try different blocking agents to reduce background

  • For phospho-antibodies, ensure rapid sample processing with phosphatase inhibitors

Sample preparation issues:

• Cell lysis optimization:

  • Test multiple lysis buffers (RIPA, NP-40, Triton X-100)

  • Include protease and phosphatase inhibitor cocktails

  • Minimize freeze-thaw cycles of samples

• Protein denaturation:

  • For Western blot, compare reducing vs. non-reducing conditions

  • Test different denaturation temperatures

  • For conformational epitopes, try native conditions

Technical considerations:

• Signaling dynamics:

  • Ensure precise timing of stimulation

  • Check cell viability before stimulation

  • Control temperature during all steps (especially important for PLAID research)

• Cell type variations:

  • PLCG2 effects may differ between cell types

  • Verify expression levels in your specific cell system

  • Consider genetic background effects

• Antibody cross-reactivity:

  • Test specificity against related proteins (e.g., PLCG1)

  • Perform peptide competition assays

  • For phospho-antibodies, validate with phosphatase treatment

These troubleshooting steps have helped researchers resolve inconsistencies in PLCG2 antibody experiments, particularly when working with novel variants or complex signaling pathways .

How can researchers optimize protocols for studying PLCG2 phosphorylation in primary immune cells?

Optimizing PLCG2 phosphorylation detection in primary immune cells requires attention to several critical factors:

Sample collection and processing:

• Minimize time between collection and fixation:

  • Process samples within 60 minutes of collection

  • Keep samples at physiological temperature until fixation

  • For blood samples, use heparin (not EDTA) as anticoagulant

• Stimulation protocol optimization:

  • For B cells: anti-IgM (5-10 μg/ml) for 1-5 minutes

  • For NK cells: IL-2 priming followed by target cell exposure

  • Include positive control stimuli (e.g., PMA/ionomycin)

  • Perform kinetic analyses to identify optimal timepoints

Phospho-epitope preservation:

• Fixation and permeabilization:

  • Fix with 1.6% paraformaldehyde for 10 minutes at 37°C

  • Permeabilize with ice-cold methanol (15 minutes at -20°C)

  • Alternative: Use commercial phospho-flow buffers (BD Phosflow, Thermo PerFix)

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

• Antibody selection:

  • Use antibodies specifically validated for phospho-flow

  • Target key phosphorylation sites: Y753, Y759, Y1197, Y1217

  • Include total PLCG2 antibody to normalize for expression levels

Analysis strategies:

• Multi-parameter analysis:

  • Include lineage markers to identify cell populations

  • Measure multiple signaling nodes simultaneously

  • Calculate phosphorylation index relative to unstimulated cells

  • Consider dimensionality reduction techniques (tSNE, UMAP) for complex datasets

• Data normalization:

  • Include standardized control samples in each experiment

  • Use stimulation index rather than raw MFI values

  • For patient samples, analyze alongside healthy controls processed identically

These optimized protocols have successfully identified phosphorylation defects in patients with PLCG2 variants, enabling accurate classification of variants and correlation with clinical phenotypes .

How can PLCG2 antibodies be used in single-cell analysis to study immune cell heterogeneity?

PLCG2 antibodies are increasingly being integrated into single-cell analysis platforms to reveal immune cell functional heterogeneity:

Single-cell technologies with PLCG2 antibody integration:

• Mass cytometry (CyTOF):

  • Metal-conjugated PLCG2 antibodies enable high-parameter analysis

  • Can simultaneously measure multiple phosphorylation sites

  • Allows correlation of PLCG2 activity with dozens of other proteins

  • Enables identification of rare cell populations with distinct signaling profiles

• Single-cell proteogenomics:

  • CITE-seq approach combining antibody detection with transcriptomics

  • Correlate PLCG2 protein levels with gene expression programs

  • Identify regulatory networks associated with PLCG2 activation

  • Link genetic variants to protein expression and function

• Imaging mass cytometry:

  • Spatial distribution of PLCG2 activation in tissue contexts

  • Visualize PLCG2 signaling within lymphoid structures

  • Map cell-cell interactions influencing PLCG2 activation

• Phospho-flow cytometry:

  • High-throughput analysis of signaling at single-cell resolution

  • Capture signaling heterogeneity within phenotypically similar cells

  • Identify differential responses to stimulation or inhibition

  • Quantify rare cell populations with aberrant signaling

These approaches have revealed unexpected heterogeneity in PLCG2 signaling among seemingly homogeneous cell populations and identified distinct signaling signatures associated with disease states like PLAID/APLAID .

What approaches can detect structural changes in PLCG2 protein conformation using antibodies?

Detecting PLCG2 conformational changes is critical for understanding activation mechanisms, particularly for gain-of-function mutations. Several antibody-based approaches can reveal these structural alterations:

• Conformation-specific antibodies:

  • Antibodies that specifically recognize active vs. inactive conformations

  • Can detect spontaneous activation in gain-of-function variants

  • Useful for monitoring the autoinhibited state disruption

• Domain-exposure assays:

  • Antibodies targeting normally hidden epitopes that become exposed upon activation

  • Particularly useful for autoinhibitory domain interactions

  • Can detect disrupted interdomain interactions in variants like D993Y

• FRET-based approaches:

  • Fluorophore-conjugated antibodies against different PLCG2 domains

  • Measure FRET efficiency as indicator of domain proximity

  • Can detect conformational changes in live cells

  • Allows real-time monitoring of activation dynamics

• Limited proteolysis combined with epitope-specific antibodies:

  • Controlled digestion reveals differential domain accessibility

  • Epitope-specific antibodies detect fragments

  • Compare proteolytic patterns between wild-type and mutant PLCG2

  • Reveals structural differences not apparent in native protein

• Cross-linking mass spectrometry with immunoprecipitation:

  • Chemical cross-linking captures domain interactions

  • Immunoprecipitation with PLCG2 antibodies enriches the protein

  • Mass spectrometry identifies cross-linked peptides

  • Maps intramolecular interactions altered in disease variants

These methods have successfully demonstrated how mutations like D993Y alter PLCG2 conformation by disrupting autoinhibitory interactions between catalytic and regulatory domains, leading to constitutive enzyme activation in APLAID syndrome .

How can PLCG2 antibodies be used to study its role in neuroinflammation and neurodegenerative diseases?

Recent research has implicated PLCG2 in neuroinflammation and neurodegenerative disorders, creating demand for specialized antibody applications in this emerging field:

Neuroimmunological applications of PLCG2 antibodies:

• Microglia-specific PLCG2 analysis:

  • Multiplex immunofluorescence combining PLCG2 with microglial markers (IBA1, TMEM119)

  • Flow cytometry of isolated microglia from brain tissue

  • Phospho-specific antibodies to assess activation state in disease models

  • Compare activation patterns in different brain regions

• Neuroinflammation assessment:

  • Track PLCG2 activation in response to amyloid-β and tau pathology

  • Co-staining with inflammatory markers to correlate with microglial activation

  • Examine relationship with TREM2 signaling pathway

  • Monitor responses to immunomodulatory therapies

• Genetic variant functional characterization:

  • Antibody-based functional studies of P522R variant (protective against Alzheimer's)

  • Compare signaling properties with antibodies against multiple phosphorylation sites

  • Assess impact on phagocytosis and inflammatory cytokine production

  • Validate findings in iPSC-derived microglia

• Brain tissue analysis:

  • Post-mortem studies comparing PLCG2 expression and activation

  • Regional variations across neurodegenerative disease progression

  • Correlation with pathological hallmarks (plaques, tangles, inflammation)

  • Co-localization with other microglial function markers

These applications help elucidate how PLCG2 signaling in microglia contributes to neuroinflammation and potentially influences neurodegenerative disease progression, opening new avenues for therapeutic development targeting this pathway .

How should researchers interpret discrepancies between phospho-PLCG2 antibody results and functional calcium flux assays?

When phospho-PLCG2 antibody results conflict with calcium flux measurements, systematic analysis is required:

Potential causes and interpretations:

• Temporal dynamics discrepancies:

  • PLCG2 phosphorylation typically peaks 1-5 minutes after stimulation

  • Calcium flux occurs within seconds and may resolve before peak phosphorylation

  • Solution: Perform detailed time-course with both readouts simultaneously

  • Interpretation: Different kinetics rather than contradictory results

• Threshold effects:

  • Small changes in phosphorylation may produce large calcium responses

  • Non-linear relationship between phosphorylation and functional output

  • Solution: Dose-response studies with quantitative analysis of both parameters

  • Interpretation: System may operate at different points on response curve

• Alternative pathway activation:

  • Calcium flux may occur through PLCG2-independent mechanisms

  • IP3 receptor sensitization can amplify small PLCG2 signals

  • Solution: Use IP3 receptor blockers or PLCG2 inhibitors to assess contribution

  • Interpretation: Multiple pathways contribute to observed calcium response

• Cell type-specific factors:

  • Buffering capacity and calcium channel expression vary between cell types

  • Signaling thresholds differ in primary cells versus cell lines

  • Solution: Compare multiple cell models and primary cells

  • Interpretation: Context-dependent signaling relationships

• Technical considerations:

  • Antibody may recognize only subset of phosphorylated forms

  • Calcium dye loading efficiency introduces variability

  • Solution: Use multiple antibodies against different phospho-sites

  • Interpretation: Methodological limitations rather than biological discrepancy

These analytical approaches have helped researchers resolve apparent contradictions between phosphorylation data and functional outcomes in studies of PLCG2 variants associated with immune dysregulation .

What are the key considerations when using PLCG2 antibodies for longitudinal studies of patient samples?

Longitudinal studies using PLCG2 antibodies require special attention to ensure comparable results across timepoints:

Critical factors for consistent longitudinal analysis:

• Sample standardization:

  • Process samples within consistent timeframe after collection

  • Standardize anticoagulant and storage conditions

  • Use identical cell isolation protocols across timepoints

  • Include reference control samples in each experimental batch

• Antibody lot consistency:

  • Purchase sufficient antibody from single lot for entire study

  • Alternatively, perform lot-to-lot normalization with reference samples

  • Document detailed antibody information for each experiment

  • Maintain consistent antibody storage conditions

• Stimulation protocol standardization:

  • Prepare master stock of stimulation reagents

  • Use consistent stimulation strength and duration

  • Control temperature precisely during stimulation

  • Document exact timing between steps

• Instrumentation calibration:

  • For flow cytometry, use calibration beads at each session

  • Document PMT voltages and compensation matrices

  • For Western blot, include standard curve samples

  • For microscopy, use reference slides for intensity calibration

• Data normalization strategies:

  • Include internal control samples at each timepoint

  • Calculate fold change rather than absolute values

  • Consider using stimulation index (stimulated/unstimulated)

  • For flow cytometry, convert to molecules of equivalent soluble fluorochrome (MESF)

• Clinical correlation:

  • Document concurrent medications and clinical activity

  • Account for potential confounding factors (infections, procedures)

  • Consider seasonal variations in immune parameters

  • Correlate with other laboratory values (e.g., immunoglobulin levels)

These approaches have enabled successful longitudinal tracking of PLCG2 signaling in patients with PLAID/APLAID during disease flares and treatment interventions .

How can researchers distinguish between primary PLCG2 defects and secondary dysregulation in complex immune disorders?

Differentiating primary PLCG2 abnormalities from secondary effects requires a multi-faceted approach:

Differential diagnostic strategies:

• Genetic-functional correlation:

  • Confirm presence of PLCG2 variant with sequencing

  • Test variant's functional impact in isolation using cell models

  • Compare patient cells with engineered cell lines expressing the variant

  • Assess if functional phenotype segregates with variant in family studies

• Pathway dissection:

  • Examine upstream regulators of PLCG2 (Syk, Btk, BLNK)

  • Test multiple downstream effectors (calcium, ERK, NFAT, NF-κB)

  • Use inhibitors to block specific pathway components

  • Determine if abnormality is restricted to PLCG2-dependent pathways

• Cell type specificity:

  • Compare PLCG2 signaling across multiple immune cell types

  • Assess if abnormality is present in all PLCG2-expressing cells

  • Pattern of cell-specific effects may distinguish primary from secondary effects

  • NK cell abnormalities are particularly informative for PLCG2 defects

• Kinetic analysis:

  • Primary PLCG2 defects typically affect early signaling events

  • Secondary dysregulation may involve compensatory changes

  • Time-course experiments can distinguish immediate from delayed effects

  • Recovery kinetics after stimulation may differ

• Response to targeted therapy:

  • Primary PLCG2 defects may respond to PLC inhibitors or downstream blockers

  • Secondary dysregulation might respond to broader immunomodulation

  • Therapeutic response pattern can provide diagnostic insights

  • Ex vivo drug testing can predict clinical responses