VIPR2 Antibody, HRP conjugated

What is VIPR2 and why is it significant in research?

VIPR2, also known as VPAC2, is a Class II G-protein coupled receptor that binds vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) with high affinity. This receptor is expressed in multiple tissues including the uterus, prostate, gastrointestinal smooth muscle, seminal vesicles, skin, blood vessels, and thymus, as well as various brain regions including the thalamus . VIPR2 is significant in research because it plays crucial roles in cell signaling pathways that influence cell migration, proliferation, and survival, particularly in cancer contexts. Recent studies have demonstrated that VIPR2 regulates cancer cell migration through specific signaling pathways .

Class II GPCRs like VIPR2 possess large N-terminal extracellular domains containing 10 highly conserved amino acids (including six cysteines), putative N-terminal leader sequences, and several potential N-glycosylation sites . This structural complexity makes VIPR2 an important but challenging target for antibody-based research.

What does HRP conjugation mean for VIPR2 antibodies, and how does it impact experimental design?

HRP (horseradish peroxidase) conjugation refers to the covalent attachment of the HRP enzyme to the VIPR2 antibody molecule. This conjugation enables direct detection in immunoassays without requiring a secondary antibody. In the case of VIPR2 antibodies like ABIN7175395, HRP conjugation specifically supports enhanced detection sensitivity in ELISA applications .

When designing experiments with HRP-conjugated VIPR2 antibodies, researchers should consider:

• Substrate compatibility: HRP-conjugated antibodies require appropriate substrates (TMB, DAB, or chemiluminescent reagents) for signal development

• Reduced incubation steps: Direct detection eliminates secondary antibody incubation, shortening protocols

• Signal amplification considerations: HRP provides enzymatic signal amplification but may not be suitable for all detection systems

• Storage requirements: HRP conjugates typically require protection from light and storage at 2-8°C with appropriate preservatives to maintain enzyme activity

For quantitative applications, standard curves should be prepared using purified VIPR2 protein at concentrations ranging from 0.1-1000 ng/mL to ensure accurate quantification within the linear range of detection.

How can I confirm the specificity of my VIPR2 antibody for immunodetection methods?

Confirming antibody specificity is critical for ensuring reliable results when working with VIPR2. A comprehensive approach includes:

Blocking peptide validation: Pre-incubate your antibody with a specific blocking peptide corresponding to the immunogen. For example, when working with antibodies targeting the extracellular domain (aa 25-37), use a VPAC2/VIPR2 extracellular blocking peptide to confirm specificity. Western blot analysis comparing samples with and without peptide pre-incubation should show signal elimination in blocked samples, as demonstrated with VPAC2 detection in rat brain, mouse brain, and human melanoma cell lysates .

Multiple tissue/sample testing: Verify detection across samples with known VIPR2 expression levels. For instance, test human, rat, and mouse samples as appropriate for your antibody's reported cross-reactivity . This multi-sample approach helps confirm the antibody can detect the target across different expression contexts.

Knockout/knockdown controls: Implement siRNA knockdown of VIPR2 as a negative control. Western blotting comparing knockdown and normal samples should demonstrate reduced signal in knockdown samples, particularly when examining downstream effects like AKT phosphorylation .

Multiple detection methods: Cross-validate using different techniques. For instance, if using a VIPR2 antibody for Western blotting, confirm specificity through complementary methods like flow cytometry or immunocytochemistry when possible .

What are the recommended protocols for using HRP-conjugated VIPR2 antibodies in ELISA?

For optimal ELISA performance with HRP-conjugated VIPR2 antibodies such as ABIN7175395 , follow these methodological guidelines:

Protocol optimization table for ELISA using HRP-conjugated VIPR2 antibodies:

When developing sandwich ELISA protocols, use antibodies targeting different epitopes of VIPR2. For instance, pair antibodies recognizing amino acids 24-126 with those targeting amino acids 81-170 to avoid epitope competition .

How should I optimize Western blot protocols specifically for VIPR2 detection?

VIPR2 detection by Western blotting requires careful optimization due to its nature as a membrane-bound receptor. Based on validated applications of VIPR2 antibodies, the following protocol adjustments are recommended:

Sample preparation:

• Use RIPA buffer supplemented with protease inhibitors for efficient extraction from membrane fractions

• For cell lines like MDA-MB-231, include phosphatase inhibitors when studying phosphorylation events downstream of VIPR2 activation

• Heat samples at 37°C instead of boiling to prevent aggregation of this membrane protein

Gel electrophoresis and transfer:

• Use 8-10% polyacrylamide gels to effectively resolve VIPR2 (approximate molecular weight 52-57 kDa)

• Transfer to PVDF membranes at 25V overnight at 4°C for complete transfer of membrane proteins

Detection optimization:

• Dilute primary antibodies (e.g., ABIN726170) at 1:500 to 1:1000 in 5% BSA/TBST

• For HRP-conjugated antibodies, extend washing steps (5 × 5 minutes) to reduce background

• Include positive controls such as brain lysates from rat or mouse, or human melanoma cell lysates as validated detection samples

Researchers should verify detection specificity using blocking peptides corresponding to the specific epitope recognized by the antibody, as demonstrated in the western blot analysis of rat brain, mouse brain, and human melanoma cell lysates .

How can I effectively use VIPR2 antibodies for studying receptor activation and downstream signaling?

VIPR2 antibodies can be powerful tools for investigating receptor activation and signaling cascade effects. Based on research applications , follow these methodological approaches:

Activation assay methodology:

• Starve cells (e.g., MDA-MB-231) for 3 hours in serum-free medium to reduce baseline signaling

• Stimulate with VIP at various concentrations (0-100 nM) for 10 minutes to activate the receptor

• Rapidly lyse cells in buffer containing phosphatase inhibitors to preserve phosphorylation states

• Analyze lysates by Western blotting using antibodies against:

  • Phosphorylated AKT (Thr308 and Ser473)

  • Total AKT protein

  • VIPR2 to confirm receptor expression levels

Data analysis approach:

• Normalize phosphorylated protein signals to total protein levels

• Present data as relative fold change compared to unstimulated controls

• Apply appropriate statistical tests (e.g., Kruskal–Wallis followed by Dunn's multiple comparison) to assess significance

Validation through genetic manipulation:

• Use siRNA-mediated knockdown of VIPR2 to confirm specificity of signaling responses

• Create stable VIPR2-overexpressing cell lines (e.g., VIPR2-EGFP) to study enhanced receptor activity

This approach has successfully demonstrated that VIP stimulation (100 nM) leads to significant AKT phosphorylation at Thr308, which is abrogated in VIPR2 knockdown cells, confirming the receptor's role in this signaling pathway .

How can VIPR2 antibodies be utilized to investigate cancer cell migration mechanisms?

Recent research has established that VIPR2 plays a critical role in cancer cell migration. When designing studies to investigate this function, researchers should consider the following methodological approach:

Experimental framework for investigating VIPR2 in cell migration:

• Establish cellular models with different VIPR2 expression levels:

  • Knockdown VIPR2 using targeted siRNA

  • Overexpress VIPR2 using stable transfection (e.g., VIPR2-EGFP constructs)

  • Use wild-type cells as controls

• Assess migration using complementary techniques:

  • Wound healing assays (2D migration)

  • Transwell migration assays (directional migration)

  • Time-lapse microscopy for real-time monitoring

• Monitor VIPR2-dependent signaling pathways:

  • Evaluate AKT phosphorylation status (Thr308 and Ser473) using Western blotting

  • Quantify results by normalizing phospho-AKT to total AKT protein levels

Research has demonstrated that VIP stimulation (100 nM) significantly increases phosphorylation of AKT at Thr308 in MDA-MB-231 cells, which correlates with enhanced migratory capacity. This effect is specifically mediated through VIPR2, as confirmed by siRNA knockdown experiments that abolished both AKT phosphorylation and migratory responses .

What are the established protocols for using VIPR2 antibodies in immunocytochemistry and flow cytometry?

Based on validated applications of VIPR2 antibodies, the following protocols have proven effective for cellular localization and quantification studies:

Immunocytochemistry protocol for VIPR2 detection:

• Culture cells on coverslips or in chamber slides to 70-80% confluence

• For membrane receptor detection, avoid permeabilization to assess only surface expression

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Block with 5% normal serum (from the same species as secondary antibody) for 1 hour

• Incubate with anti-VIPR2 antibody at 1:50 dilution (e.g., extracellular targeting antibody AVR-002)

• Visualize using appropriate fluorophore-conjugated secondary antibody (e.g., goat anti-rabbit-AlexaFluor-594)

• Counterstain nuclei with DAPI and mount for imaging

Flow cytometry protocol for cell surface VIPR2 detection:

• Harvest live cells using enzyme-free dissociation buffer to preserve surface epitopes

• Wash cells in cold PBS containing 2% FBS (FACS buffer)

• Incubate 1×10⁶ cells with anti-VIPR2 antibody (2.5μg) for 30 minutes on ice

• Wash three times with FACS buffer

• Incubate with fluorophore-conjugated secondary antibody (e.g., goat-anti-rabbit-APC)

• Analyze using appropriate flow cytometer channels

This approach has been successfully demonstrated for detecting VIPR2 expression in human Jurkat T-cell leukemia cells and rat PC12 pheochromocytoma cells , confirming the cell surface localization of the receptor.

How do I design rigorous controls when investigating VIPR2 function in experimental systems?

Robust experimental design requires comprehensive controls to validate findings related to VIPR2 function:

Essential controls for VIPR2 research:

Implementing these controls has enabled researchers to conclusively demonstrate that observed effects on AKT phosphorylation and cell migration are specifically mediated through VIPR2 rather than other related receptors or non-specific interactions .

What are common issues when using HRP-conjugated VIPR2 antibodies and how can they be resolved?

Researchers working with HRP-conjugated VIPR2 antibodies may encounter several technical challenges. Here are evidence-based solutions to address common issues:

High background signal:

• Cause: Insufficient blocking or excessive antibody concentration

• Solution: Increase blocking time to 2 hours with 5% BSA in PBS; optimize antibody dilution through titration experiments starting at 1:1000 and testing up to 1:5000; extend washing steps to 5 × 5 minutes with 0.1% PBST

Weak or absent signal:

• Cause: Inadequate antigen retrieval (for tissue sections) or low target expression

• Solution: For tissue sections, optimize antigen retrieval methods; confirm VIPR2 expression in your sample type (known high expressors include brain tissue and melanoma cell lines like Malme-3M) ; for cell lines, consider VIP stimulation to upregulate receptor expression

Non-specific bands in Western blotting:

• Cause: Cross-reactivity with related receptors (e.g., VIPR1/VPAC1)

• Solution: Validate with blocking peptide specific to the antibody's target epitope; compare signal pattern with protein samples from VIPR2 knockdown models

Inconsistent results between experiments:

• Cause: Antibody degradation or variability in experimental conditions

• Solution: Aliquot antibodies upon receipt to minimize freeze-thaw cycles; standardize protein concentration across experiments; include positive control samples (e.g., rat brain lysate) in each experiment

How should I approach quantitative analysis when using VIPR2 antibodies in different applications?

Accurate quantification requires appropriate methodological approaches depending on the technique employed:

Western blot quantification:

• Use standard curve of recombinant VIPR2 protein (10-100 ng) for absolute quantification

• For relative quantification, normalize VIPR2 signal to housekeeping proteins like β-actin

• For phosphorylation studies, calculate the ratio of phosphorylated to total protein (e.g., pAKT/AKT) as demonstrated in VIP stimulation experiments

• Use densitometry software with background subtraction and report values as fold-change relative to control

ELISA quantification:

• Generate standard curves using purified recombinant VIPR2 (0.1-1000 ng/mL)

• Ensure samples fall within the linear range of the standard curve (typically 1-100 ng/mL)

• Run technical triplicates and report the mean ± standard deviation

• Include spike-in recovery controls to validate accuracy in complex matrices

Flow cytometry quantification:

• Report median fluorescence intensity rather than mean (less affected by outliers)

• Use antibody binding capacity (ABC) beads to convert arbitrary units to molecules per cell

• Include fluorescence-minus-one (FMO) controls to set accurate gates

• For comparing expression levels between samples, use consistent instrument settings and calibration

These approaches enable precise quantification of VIPR2 expression levels and activation states as demonstrated in studies examining VIPR2-dependent AKT phosphorylation .

How are VIPR2 antibodies being applied in current cancer research?

Recent advances in VIPR2 research have revealed significant implications for cancer biology:

Current research applications of VIPR2 antibodies in oncology:

• Investigation of migration mechanisms: Studies have conclusively demonstrated that VIPR2 regulates cancer cell migration through AKT signaling pathways. This finding suggests potential therapeutic approaches targeting this receptor in metastatic disease .

• Signaling pathway elucidation: VIP stimulation (100 nM) of MDA-MB-231 breast cancer cells induces significant AKT phosphorylation at Thr308, with this effect being specifically mediated through VIPR2 rather than other VIP receptors .

• Expression profiling across tumor types: Immunohistochemical detection of VIPR2 in various cancers is establishing expression patterns that correlate with clinical outcomes. Studies have detected VIPR2 in melanoma cell lines (e.g., Malme-3M), suggesting potential roles in skin cancers .

• Receptor-targeted therapeutic development: Characterization of the extracellular domain (amino acids 25-37) using specific antibodies is facilitating the development of receptor antagonists and targeted therapies .

These research directions highlight how VIPR2 antibodies are advancing understanding of cancer biology beyond simple detection, providing insights into functional mechanisms that may guide therapeutic strategies.

What methodological considerations are important when using VIPR2 antibodies in multi-parameter analysis?

As research complexity increases, integrating VIPR2 detection with other parameters requires careful methodological planning:

Multi-parameter experimental design guidelines:

• Antibody panel design for flow cytometry:

  • Select VIPR2 antibodies with compatible fluorophores for multi-color panels

  • Consider brightness hierarchy (assign brightest fluorophores to lowest-expressed targets)

  • Include compensation controls for each fluorophore

  • Validate that antibody binding is not affected by fixation procedures when combining with intracellular markers

• Co-localization studies in microscopy:

  • When combining VIPR2 detection with other proteins of interest:

    • Use primary antibodies from different host species to prevent cross-reactivity

    • Validate antibody specificity independently before combination

    • Employ appropriate controls including single-stained samples

• Multiplexed signaling analysis:

  • When studying VIPR2-mediated signaling networks:

    • Include time-course experiments (0-60 minutes post-stimulation)

    • Analyze multiple phosphorylation sites simultaneously

    • Consider phospho-protein arrays or multiplex Western blotting

    • Use pathway inhibitors to establish signaling hierarchies

This comprehensive approach has enabled researchers to establish that VIPR2 activation leads to AKT phosphorylation and subsequent effects on cell migration, placing this receptor within broader cellular signaling networks relevant to cancer biology .

What are promising directions for advancing VIPR2 antibody applications in research?

Based on current research findings and technological trends, several promising directions for VIPR2 antibody applications are emerging:

• Development of conformational state-specific antibodies: New antibodies that specifically recognize active versus inactive conformations of VIPR2 could provide valuable tools for studying receptor dynamics and activation mechanisms in real-time.

• Integration with proximity labeling techniques: Combining VIPR2 antibodies with BioID or APEX2 proximity labeling would enable comprehensive mapping of VIPR2 interactomes in different cellular contexts and activation states.

• Single-cell analysis of VIPR2 expression and signaling: Applying VIPR2 antibodies in single-cell technologies could reveal heterogeneity in receptor expression and downstream signaling across cell populations, particularly in tumor microenvironments.

• Therapeutic applications: Beyond research tools, VIPR2 antibodies modified as blocking antibodies could have therapeutic potential in conditions where VIP-VIPR2 signaling contributes to pathology, such as cancer cell migration .