VAV3 Antibody, HRP conjugated

What is VAV3 and what cellular functions does it mediate?

VAV3 functions as an exchange factor for GTP-binding proteins including RhoA, RhoG and, to a lesser extent, Rac1. It physically binds to the nucleotide-free states of these GTPases and plays critical roles in angiogenesis, with its recruitment by phosphorylated EPHA2 being essential for EFNA1-induced RAC1 GTPase activation and vascular endothelial cell migration and assembly. VAV3 is also important for integrin-mediated signaling in certain cell types, particularly in osteoclasts where it's required for proper cytoskeleton organization and function through integrin alpha-v/beta-1 signaling. Additionally, VAV3 facilitates wound healing processes and macrophage phagocytosis of apoptotic neutrophils .

What applications are VAV3 Antibody, HRP conjugated suitable for?

VAV3 Antibody, HRP conjugated is specifically validated for ELISA, immunohistochemistry of paraffin-embedded tissues (IHC-P), and immunohistochemistry of frozen sections (IHC-F). The direct horseradish peroxidase conjugation eliminates the need for secondary antibody incubation, simplifying experimental workflows and potentially reducing background issues in these applications .

What are the recommended dilutions for different applications?

Based on validated protocols, the following dilution ranges are recommended for optimal results with VAV3 Antibody, HRP conjugated:

*Note: While the HRP-conjugated form is not specifically validated for Western blot in the provided data, the unconjugated form uses these dilutions and may serve as a starting point for optimization .

What species reactivity has been confirmed for VAV3 Antibody, HRP conjugated?

The antibody has confirmed reactivity with human and rat samples. Based on sequence homology analysis, it's also predicted to recognize VAV3 from mouse, dog, cow, horse, and rabbit samples, though these cross-reactivities would need experimental validation for definitive confirmation .

How should VAV3 Antibody, HRP conjugated be stored to maintain activity?

The antibody should be stored at -20°C in its original buffer containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol. To avoid activity loss from repeated freeze-thaw cycles, it's recommended to aliquot the antibody into multiple smaller volumes upon receipt. Proper storage ensures the maintenance of binding capacity and signal generation for accurate experimental results .

What controls should be included when using VAV3 Antibody, HRP conjugated?

Proper experimental design requires several controls:

• Negative control: Samples known to lack VAV3 expression or VAV3 knockout cell lines (as demonstrated in )

• Isotype control: Using an irrelevant HRP-conjugated IgG from the same host species (rabbit)

• Loading control: For Western blot applications, probing for housekeeping proteins like GAPDH

• Blocking peptide control: Pre-incubation of the antibody with the immunizing peptide to demonstrate binding specificity .

What is the immunogen used to generate this antibody and how does it affect epitope recognition?

The VAV3 Antibody, HRP conjugated is generated using a KLH-conjugated synthetic peptide derived from mouse VAV3, specifically targeting the region between amino acids 701-800 of the 847 amino acid protein. This C-terminal epitope recognition is important to consider when designing experiments, particularly if studying truncated forms of VAV3 or when protein interactions might mask this region .

How can VAV3 Antibody, HRP conjugated be utilized to investigate VAV3's role in cell cycle regulation?

Research has demonstrated that VAV3 expression levels vary dramatically during different phases of the cell cycle, with particularly elevated expression during mitosis compared to interphase. To investigate this phenomenon, researchers can:

• Synchronize cells using techniques like double thymidine block or nocodazole treatment

• Collect cells at defined time points representing different cell cycle phases

• Perform Western blot analysis using VAV3 Antibody, HRP conjugated to quantify expression changes

• Correlate VAV3 expression with cell cycle markers and mitotic indices

• Complement protein analysis with immunofluorescence to visualize subcellular localization changes

This approach allows for tracking VAV3 dynamics throughout cell division, potentially revealing regulatory mechanisms controlling its expression and activity during mitosis .

What methodological approaches can identify VAV3's nuclear functions in hematological malignancies?

Recent discoveries have revealed critical nuclear functions for VAV3 in B-cell lymphoblastic leukemia, particularly in relation to polycomb repression complex-1 (PRC1) activity. To investigate these functions:

• Perform subcellular fractionation to separate nuclear and cytoplasmic compartments

• Use VAV3 Antibody, HRP conjugated for Western blot detection of VAV3 in different cellular fractions

• Conduct co-immunoprecipitation experiments to identify interactions between VAV3 and PRC1 components (BMI1, Ring1B)

• Apply chromatin immunoprecipitation (ChIP) to determine genomic binding sites of VAV3-associated complexes

• Analyze H2AK119 mono-ubiquitination patterns (mediated by PRC1) in relation to VAV3 expression/localization

These approaches help establish the mechanistic role of nuclear VAV3 in transcriptional regulation via polycomb complex activity, particularly relevant in BCR-ABL-positive leukemias .

How can researchers quantitatively assess the impact of VAV3 on GTPase activation?

To measure VAV3's guanine nucleotide exchange factor (GEF) activity toward its target GTPases:

• Perform pull-down assays using GST-fused effector domains that specifically bind activated (GTP-bound) forms of Rac1, RhoA, or RhoG

• Compare GTPase activation levels in cells with normal vs. altered VAV3 expression

• Use VAV3 Antibody, HRP conjugated to confirm VAV3 expression levels in the experimental system

• Complement biochemical assays with fluorescence-based GTPase activity sensors

• Correlate GTPase activation with downstream cellular phenotypes like cytoskeletal reorganization

This multi-faceted approach allows researchers to establish quantitative relationships between VAV3 expression/activity and the activation state of its downstream effectors .

What are potential causes of high background when using VAV3 Antibody, HRP conjugated in immunohistochemistry?

High background signals can significantly impact the interpretation of immunohistochemistry results. Common causes and solutions include:

• Insufficient blocking: Extend blocking time with 1-5% BSA or serum

• Excessive antibody concentration: Further dilute the primary antibody beyond the recommended 1:200-400 range

• Endogenous peroxidase activity: Add additional peroxidase quenching step with hydrogen peroxide

• Nonspecific binding: Include 0.1-0.3% Triton X-100 in wash buffers

• Tissue fixation issues: Optimize fixation protocol and antigen retrieval methods

Systematic optimization of these parameters can significantly improve signal-to-noise ratios and enable clear visualization of specific VAV3 expression patterns .

How can researchers verify the specificity of VAV3 Antibody, HRP conjugated?

Antibody specificity validation is critical for reliable interpretations. Methods include:

• Testing with VAV3 knockout cell lines, which should show complete absence of signal (as demonstrated for other VAV3 antibodies in )

• Peptide competition assays, where pre-incubation with the immunizing peptide should abolish specific binding

• Comparison of staining patterns with alternative antibodies targeting different VAV3 epitopes

• Correlation of protein detection with mRNA expression data

• Western blot analysis to confirm detection of bands at the expected molecular weight (98 kDa for VAV3)

This multi-method validation approach ensures that experimental observations are truly attributable to VAV3 and not to non-specific antibody interactions .

What steps should be taken when VAV3 Antibody, HRP conjugated produces weaker-than-expected signals?

Signal optimization strategies include:

• Antibody concentration adjustment: Try reducing dilution factors to increase antibody concentration

• Extended incubation times: Increase primary antibody incubation from standard protocols to overnight at 4°C

• Enhanced detection systems: Use amplification systems like tyramide signal amplification (TSA)

• Antigen retrieval optimization: Test different antigen retrieval buffers and conditions

• Sample preparation refinement: Ensure proteins aren't degraded during sample collection and processing

These approaches can help recover signal when working with samples having low VAV3 expression levels or when dealing with partially degraded samples .

How should researchers interpret VAV3 localization differences between normal and malignant cells?

Research has shown that VAV3 localization differs significantly between normal and malignant cells, particularly in B-cell malignancies. When analyzing such differences:

• Compare nuclear-to-cytoplasmic ratios of VAV3 staining between normal and malignant samples

• Correlate subcellular localization with activation markers like phosphorylation status

• Analyze co-localization with known interaction partners in different cellular compartments

• Consider cell cycle phase when interpreting localization data, as VAV3 expression varies throughout the cell cycle

• Examine relationships between localization patterns and disease progression markers

The nuclear accumulation of VAV3 in B-cell leukemias appears functionally significant, as it contributes to polycomb repression complex-1 activity, suggesting that localization data may have both mechanistic and potential prognostic value .

What considerations are important when analyzing VAV3 expression in relation to integrin signaling?

VAV3 plays critical roles in integrin-mediated signaling, particularly through:

• Integrin αvβ1 signaling in osteoclasts, affecting cytoskeletal organization

• Integrin β2-mediated macrophage adhesion

• Integrin β3-mediated adhesion (to a lesser extent)

• Notably, VAV3 does not significantly affect integrin β1-mediated adhesion

When analyzing VAV3 in relation to integrin function, researchers should:

• Examine co-localization with specific integrin subunits in cellular adhesion structures

• Analyze downstream RAC1 activation in relation to VAV3 and integrin co-expression

• Consider cell-type specificity of the VAV3-integrin relationship

• Evaluate potential compensation by other VAV family members (VAV1, VAV2)

This approach helps dissect the specific contribution of VAV3 to distinct integrin-mediated cellular processes .

How should contradictory VAV3 expression data across different experimental systems be reconciled?

When faced with conflicting data regarding VAV3 expression or function:

• Consider cell-type specific differences in VAV3 regulation and function

• Evaluate the impact of different experimental conditions (growth factors, cell density, etc.)

• Analyze the specific VAV3 domains/regions detected by different antibodies

• Assess potential technical variables like antibody lot-to-lot variation

• Examine post-translational modifications that might affect antibody binding

The biological context significantly influences VAV3 expression and function - for instance, VAV3 shows dramatically different expression levels between interphase and mitotic cells. These context-dependent variations should be systematically analyzed before concluding that data is truly contradictory rather than revealing biological complexity .