ADGRB2 Antibody, HRP conjugated

What is ADGRB2 and why is it important in research?

ADGRB2 (Adhesion G Protein-Coupled Receptor B2), also known as BAI2 (Brain-specific Angiogenesis Inhibitor 2), belongs to the ADGRB subfamily of adhesion GPCRs, which are primarily expressed in brain tissue. This receptor is important in neuroscience research because mutations have been associated with neurological disorders, including a de novo C-terminal mutation (R1465W) identified in a patient with progressive spastic paraparesis and other neurological symptoms . Unlike its better-characterized family members BAI1/ADGRB1 and BAI3/ADGRB3 which have established roles at synapses, BAI2's precise functions are still being elucidated. Studies in mice lacking BAI2 have shown increased hippocampal neurogenesis and resilience to learned-helplessness behavior, suggesting roles in neuroplasticity and mood regulation . The receptor's activation mechanism involves N-terminal cleavage, representing a common feature of adhesion GPCRs that is critical for understanding their function.

What types of ADGRB2 antibodies are available for research applications?

Several types of ADGRB2 antibodies are available for research, each with specific applications:

• C-terminal targeted antibodies (e.g., Mab Technologies, cat. #BAI2-3) that recognize the intracellular domain of the receptor

• N-terminal targeted antibodies that bind to the extracellular region

• Domain-specific antibodies that recognize particular functional regions

• Tag-specific antibodies for detecting epitope-tagged versions of ADGRB2 in transfection studies

• HRP-conjugated antibodies that provide direct enzymatic detection capabilities

Researchers should select the appropriate antibody based on their experimental design, considering factors such as the cellular location of the epitope (extracellular vs. intracellular), the native conformation of the protein, and the detection method being employed. For studies involving the R1465W mutation, C-terminal antibodies may provide valuable insights since this mutation occurs in the C-terminal region of the receptor .

How can I validate the specificity of ADGRB2 antibodies?

Validation of ADGRB2 antibodies is crucial as GPCRs present particular challenges for antibody specificity. According to research examining multiple GPCR antibodies, "lack of selectivity appears to be the rule rather than the exception for antibodies against G-protein-coupled and perhaps also other receptors" . The following methodological approaches are recommended for proper validation:

• Test in knockout/knockdown systems: The most definitive validation involves testing the antibody in ADGRB2 knockout tissue or cells with ADGRB2 knockdown via siRNA

• Differential expression systems: Compare antibody reactivity in cells overexpressing ADGRB2 versus non-transfected controls

• Multiple epitope concordance: Use multiple antibodies targeting different ADGRB2 epitopes and verify similar staining patterns

• Cross-reactivity assessment: Test against related proteins (BAI1/ADGRB1, BAI3/ADGRB3) expressed in the same system

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight for ADGRB2

Importantly, the practice of only using blocking peptide controls (pre-incubating the antibody with the immunizing peptide) is insufficient for demonstrating specificity . Comprehensive validation using multiple approaches provides greater confidence in antibody specificity and experimental results.

What are the optimal conditions for using HRP-conjugated ADGRB2 antibodies in Western blots?

Based on published protocols for ADGRB2/BAI2 detection, the following optimized conditions are recommended for Western blotting with HRP-conjugated ADGRB2 antibodies:

Sample preparation:

• Lyse cells or tissues in 1% Triton X-100 buffer containing protease inhibitors and phosphatase inhibitors

• For membrane proteins like ADGRB2, solubilization is critical for extraction

• Reduce samples in Laemmli buffer before loading

Gel electrophoresis and transfer:

• Use 4-20% Tris-glycine gradient gels for optimal separation of the large ADGRB2 protein (~170-180 kDa)

• Transfer to nitrocellulose membranes at low voltage (30V) overnight at 4°C for better transfer of large proteins

• Verify transfer efficiency with a reversible protein stain

Blocking and antibody incubation:

• Block with 5% milk in buffer containing 50mM NaCl, 10mM HEPES pH 7.3, 0.1% Tween-20

• For HRP-conjugated antibodies, dilute according to manufacturer's recommendation (typically 1:1000 to 1:5000)

• Incubate for 1 hr at room temperature or overnight at 4°C

• Wash thoroughly (3-5 times for 5-10 minutes each) with TBST or PBST buffer

Detection:

• Use enhanced chemiluminescence (ECL) substrate compatible with HRP

• For quantitative analysis, use a digital imager like Li-Cor Odyssey Imager

• Optimize exposure time to avoid signal saturation for accurate quantification

These conditions should be optimized for each specific HRP-conjugated ADGRB2 antibody and research application.

How can I design experiments to study the R1465W mutation in ADGRB2?

The R1465W mutation in ADGRB2 provides a valuable model for understanding receptor function and disease mechanisms. Based on published research, the following experimental approaches can be employed:

Comparative signaling studies:

• Generate expression constructs for wild-type and R1465W mutant ADGRB2

• Create N-terminally truncated constructs (ADGRB2ΔNT) to mimic the activated form of the receptor

• Transfect into appropriate cell lines (HEK293, COS-7)

• Measure downstream signaling pathways (cAMP, Ca2+, ERK)

• Perform co-immunoprecipitation with different G proteins to assess coupling preferences

Trafficking analysis:

• Compare surface expression using biotinylation assays with streptavidin precipitation followed by Western blotting with ADGRB2 antibodies

• Implement pulse-chase experiments to assess protein stability and turnover rates

• Perform immunofluorescence studies to visualize receptor distribution

Protein interaction studies:

• Identify interaction partners through co-immunoprecipitation followed by mass spectrometry

• Compare wild-type and R1465W binding to known partners (β-arrestins, endophilin A1)

• Map interaction domains using truncation mutants or peptide arrays

Structural implications:

• Use computational modeling to predict how R1465W affects receptor conformation

• Employ conformation-specific antibodies to detect structural changes

• Perform accessibility studies to determine if the mutation alters epitope exposure

Published research has already revealed that this mutation increases both total and surface expression of ADGRB2, alters G protein coupling preference (shifting from Gαz to Gαi), and disrupts interaction with endophilin A1 . These findings provide a framework for further investigations.

What controls should be included when using HRP-conjugated ADGRB2 antibodies?

Proper experimental controls are essential for generating reliable data with ADGRB2 antibodies. The following controls should be incorporated:

Specificity controls:

• Negative control: ADGRB2 knockout or knockdown samples

• Competing peptide control: Pre-incubation of antibody with immunizing peptide

• Isotype control: Non-specific antibody of same isotype and concentration

Technical controls:

• Loading control: Housekeeping protein detection (e.g., GAPDH, β-actin)

• Transfer control: Reversible total protein stain of membrane

• Enzyme activity control: For HRP-conjugated antibodies, include a known positive control

Application-specific controls:

Mutation-specific controls:
When studying the R1465W mutation, include:

• Wild-type ADGRB2 transfected cells as reference

• Empty vector transfected cells as negative control

• Related mutations in the same region to test position-specificity

The inclusion of these controls helps ensure data validity and facilitates accurate interpretation of results.

How can HRP-conjugated ADGRB2 antibodies be used to study receptor trafficking?

HRP-conjugated ADGRB2 antibodies can be powerful tools for investigating receptor trafficking dynamics through several advanced methodological approaches:

Surface biotinylation assays:
HRP-conjugated ADGRB2 antibodies can detect biotinylated receptors in cell surface fraction experiments, allowing quantification of surface versus intracellular receptor pools . The protocol typically involves:

• Biotinylating surface proteins with cell-impermeable reagents

• Isolating biotinylated proteins with streptavidin agarose

• Detecting ADGRB2 in the biotinylated fraction using HRP-conjugated antibodies

• Comparing total versus surface expression under different conditions (e.g., wild-type vs. R1465W mutant)

Internalization assays:
To study receptor internalization kinetics:

• Biotinylate surface proteins at time zero

• Allow cells to internalize receptors for varying time periods

• Strip remaining surface biotin with a membrane-impermeable reducing agent

• Isolate protected (internalized) biotinylated proteins

• Detect ADGRB2 using HRP-conjugated antibodies

Recycling studies:
By combining surface biotinylation with temperature shifts to control trafficking, researchers can quantify receptor recycling rates between endosomal compartments and the plasma membrane.

Co-trafficking with interacting proteins:
HRP-conjugated ADGRB2 antibodies can determine whether the receptor co-traffics with binding partners like endophilin A1 or β-arrestins through co-immunoprecipitation of trafficking complexes at different time points .

These approaches have revealed that the R1465W mutation increases both total and surface expression of ADGRB2, contributing to its gain-of-function phenotype .

How can I use ADGRB2 antibodies to identify and characterize novel binding partners?

ADGRB2 antibodies are valuable tools for discovering and validating protein-protein interactions using several methodological approaches:

Co-immunoprecipitation (Co-IP):

• Solubilize membrane proteins in mild detergents (1% Triton X-100)

• Immunoprecipitate ADGRB2 using specific antibodies bound to protein A/G agarose or magnetic beads

• Analyze co-precipitated proteins by Western blotting for suspected partners or mass spectrometry for unbiased discovery

This approach has successfully identified endophilin A1 as a novel binding partner for ADGRB2 . The study demonstrated that wild-type ADGRB2 interacts with endophilin A1, but this interaction is disrupted by the R1465W mutation, providing a potential mechanism for altered receptor trafficking and function.

Proximity-based labeling techniques:

• Generate fusion proteins of ADGRB2 with enzymes like BioID or APEX2

• Express these constructs in relevant cell types

• Activate the enzyme to biotinylate proteins in close proximity to ADGRB2

• Isolate biotinylated proteins and identify by mass spectrometry

• Validate hits using ADGRB2 antibodies in reverse Co-IP experiments

Peptide array mapping:

• Generate arrays of overlapping peptides covering ADGRB2 sequence

• Probe arrays with purified potential interaction partners

• Identify binding regions

• Use ADGRB2 antibodies targeting these regions to competitively disrupt interactions

These approaches can reveal how ADGRB2 participates in signaling networks and how disease-associated mutations like R1465W disrupt normal interaction patterns.

What approaches can be used to study ADGRB2 in brain tissue samples?

Working with ADGRB2 antibodies in brain tissue presents unique challenges and requires specialized methodological considerations:

Tissue preparation considerations:

• Fixation method: Different fixatives can affect epitope accessibility

• Antigen retrieval: May be necessary to unmask epitopes after fixation

• Section thickness: Thinner sections (5-10μm) usually provide better antibody penetration

• Post-mortem interval: Can affect protein integrity and post-translational modifications

Special methodological considerations:

• Background autofluorescence: Brain tissue often has high lipofuscin autofluorescence

  • Solution: Use Sudan Black B (0.1%) to quench autofluorescence

  • Alternative: Use HRP-conjugated antibodies with DAB detection

• Cross-reactivity with related proteins: BAI1/ADGRB1 and BAI3/ADGRB3 are also expressed in brain

  • Solution: Validate with knockout controls or competing peptides

  • Alternative: Use RNA probes (RNAscope) in parallel to confirm expression patterns

• Cell-type specificity: Determining which neural cell types express ADGRB2

  • Solution: Co-stain with cell-type markers (NeuN, GFAP, Iba1)

  • Approach: Use double immunofluorescence or sequential immunohistochemistry

• Subcellular localization: ADGRB2 may localize to specific neuronal compartments

  • Solution: Use super-resolution microscopy techniques

  • Alternative: Perform subcellular fractionation followed by Western blotting

Research has shown that BAI2/ADGRB2 is most abundantly expressed in brain tissue , making these considerations particularly important for neurological studies, especially those investigating the connection between ADGRB2 mutations and conditions like progressive spastic paraparesis.

How can I quantitatively analyze data obtained using ADGRB2 antibodies?

Proper quantitative analysis of ADGRB2 antibody data requires rigorous methodology:

Western blot quantification:

• Use digital imaging systems (e.g., Li-Cor Odyssey) rather than film for better linearity

• Verify signal is within linear detection range by running a dilution series

• Normalize to loading controls (housekeeping proteins or total protein stains)

• For comparing wild-type and mutant ADGRB2 (e.g., R1465W), express results as relative changes

• Perform statistical analysis on biological replicates (n≥3) rather than technical replicates

Surface expression quantification:

• For biotinylation experiments, calculate the ratio of surface to total ADGRB2

• For ELISA-based approaches, generate standard curves with recombinant proteins if available

• For immunofluorescence, measure fluorescence intensity at membrane vs. cytoplasm

• Always compare experimental conditions processed in parallel to minimize technical variation

Protein interaction analysis:

• For co-immunoprecipitation experiments, normalize the amount of co-precipitated protein to the amount of precipitated ADGRB2

• Include IgG controls to account for non-specific binding

• For comparing wild-type and R1465W effects on protein interactions (e.g., with endophilin A1), express as percent change from wild-type

Statistical considerations:

• Perform appropriate statistical tests based on data distribution (t-test, ANOVA, non-parametric tests)

• Account for multiple comparisons when appropriate

• Report both statistical significance and effect size

• Be transparent about outlier handling and exclusion criteria

These quantitative approaches have been successfully applied to demonstrate significant differences between wild-type and R1465W mutant ADGRB2 in terms of signaling activity, surface expression, and protein interactions .

What are common pitfalls in data interpretation when using ADGRB2 antibodies?

Researchers should be aware of these common pitfalls when interpreting ADGRB2 antibody data:

Specificity assumptions:

• Assuming antibody specificity without proper validation

• Relying solely on blocking peptide experiments when research shows this is insufficient

• Mistaking related proteins (BAI1/ADGRB1, BAI3/ADGRB3) for ADGRB2 due to cross-reactivity

• Failing to account for non-specific binding in complex samples like brain tissue

Quantification errors:

• Analyzing saturated signals that exceed the linear range of detection

• Improper background subtraction

• Using inappropriate normalization methods

• Over-interpreting small differences that may not be biologically significant

Overinterpretation of mutation effects:

• Assuming that in vitro findings with the R1465W mutation directly explain patient symptoms

• Not accounting for overexpression artifacts in transfection studies

• Failing to consider compensatory mechanisms present in vivo but absent in vitro

Methodological misinterpretations:

• Confusing total protein changes with altered subcellular distribution

• Misinterpreting antibody accessibility changes as actual protein level changes

• Not accounting for post-translational modifications that may affect antibody binding

To avoid these pitfalls, researchers should:

• Rigorously validate antibodies using multiple approaches

• Include all appropriate controls

• Be transparent about limitations in both experimental design and data interpretation

• Consider complementary approaches that don't rely solely on antibodies

• Be cautious when extrapolating from in vitro to in vivo contexts