ABI3 Antibody

What is ABI3 protein and why is it significant for research?

ABI3, also known as NESH (New molecule including SH3), is a 366 amino acid protein that plays crucial roles in multiple cellular functions. It has significant research value due to its essential role in phagocytic activity, which helps clear cellular debris and toxins, supporting immune response and tissue homeostasis . Additionally, ABI3 is genetically linked to Alzheimer's disease (AD), potentially contributing to microglia-mediated AD progression through regulation of microglial morphology . Its interactions with Abelson tyrosine kinase (ABL1) implicate it in signaling pathways that mediate mitogenic responses to growth factor receptor activation . Furthermore, ABI3 is involved in regulating cell growth, transformation, and modulating cell motility through actin polymerization in structures like lamellipodia and filopodia . These diverse functions make ABI3 a target of interest across multiple research fields.

How do I select the appropriate ABI3 antibody for my specific research application?

Selecting the appropriate ABI3 antibody requires careful consideration of multiple experimental parameters. First, determine your target species (human, mouse, rat) and ensure the antibody has been validated for reactivity with that species. For example, some antibodies like those from Abcam (ab214318) react with human and rat samples , while others like Boster Bio's A03909 react with human and mouse .

Second, consider your application requirements: for immunohistochemistry on paraffin-embedded tissues, Abcam's rabbit polyclonal antibody has been validated ; for Western blotting and immunoprecipitation, Cell Signaling Technology's antibody (#23060) is suitable ; for more diverse applications including immunofluorescence and ELISA, Santa Cruz's mouse monoclonal (C-7) offers broader functionality .

Finally, evaluate the specific region of ABI3 recognized by the antibody (epitope), especially if you're investigating protein interactions or post-translational modifications. For instance, Abcam's antibody targets a synthetic peptide within amino acids 100-200 of human ABI3 , which may be advantageous for certain structural or functional studies.

What are the optimal conditions for using ABI3 antibodies in Western blotting experiments?

For Western blotting with ABI3 antibodies, consider these methodological parameters for optimal results:

Sample preparation: Complete cell lysis is crucial as ABI3 interacts with cytoskeletal components. Use RIPA buffer with protease inhibitors, and sonicate briefly to ensure complete solubilization.

Dilution ratios: Based on available data, start with these recommended dilutions:

• Cell Signaling Technology #23060: 1:1000

• Boster Bio A03909: 1:500-1:2000

Expected molecular weight detection: ABI3 typically appears at 55-65 kDa . Be prepared to observe multiple bands due to potential post-translational modifications or isoforms.

Controls: Include a positive control tissue/cell line known to express ABI3. For negative controls, consider using siRNA knockdown samples or tissues from ABI3-knockout models if available.

Blocking conditions: 5% non-fat dry milk in TBST is typically sufficient, but for phospho-specific detection, 5% BSA may yield better results.

Remember that ABI3 is involved in protein-protein interactions, so gentle denaturation conditions may better preserve epitope recognition in some cases.

How should I optimize immunohistochemistry protocols for ABI3 detection in brain tissue sections?

Optimizing immunohistochemistry protocols for ABI3 detection in brain tissue requires special consideration due to the protein's role in microglial function and Alzheimer's disease pathology:

Antigen retrieval: For formalin-fixed, paraffin-embedded tissues, heat-mediated antigen retrieval using citrate buffer (pH 6.0) is recommended. Abcam's antibody has been validated for IHC-P at a dilution of 1:200 .

Tissue preparation considerations: Given ABI3's role in microglial function, preservation of microglial morphology is critical. Use brief fixation times (≤24 hours) with 4% paraformaldehyde to minimize antigenic masking.

Detection systems: DAB (3,3'-diaminobenzidine) staining provides good visualization of ABI3 expression patterns in brain tissue . For co-localization studies with other microglial markers, consider fluorescent secondary antibodies compatible with your primary antibody species (rabbit or mouse).

Counterstaining options: Light hematoxylin counterstaining helps visualize tissue architecture without obscuring ABI3 signal. In fluorescent approaches, DAPI provides nuclear context.

Controls for validation: Include positive control tissues (kidney samples have shown good reactivity) and negative controls (primary antibody omission and ideally ABI3-knockout tissue if available).

How do I troubleshoot inconsistent or weak ABI3 antibody signals in my experiments?

When encountering inconsistent or weak ABI3 antibody signals, systematically address these potential issues:

Antibody-specific factors:

• Confirm antibody viability by avoiding freeze-thaw cycles; store according to manufacturer recommendations (typically -20°C for long-term storage, 4°C for up to one month)

• Validate antibody lot performance using positive control samples

• Consider trying different antibody clones/vendors if persistent issues occur

Protocol parameters:

• For Western blotting: Increase protein loading (up to 50μg), extend primary antibody incubation (overnight at 4°C), or use more sensitive detection systems

• For IHC/IF: Optimize antigen retrieval duration, increase antibody concentration incrementally, or extend incubation times

• For IP: Increase antibody amount (up to 1:50 dilution) and extend binding time

Biological variables:

• Verify ABI3 expression levels in your experimental system using validated positive controls

• Consider tissue-specific expression patterns; ABI3 may be enriched in specific cell populations (e.g., microglia in brain tissue)

• Account for potential post-translational modifications that might mask epitopes

Technical solutions table:

How can I distinguish between specific ABI3 signals and non-specific binding in my experiments?

Distinguishing specific ABI3 signals from non-specific binding requires multiple validation approaches:

Molecular weight verification: In Western blotting, ABI3 should appear primarily at 55-65 kDa . Bands at dramatically different molecular weights may represent non-specific binding or cross-reactivity.

Knockdown/knockout controls: The gold standard for specificity validation is demonstrating signal reduction in samples where ABI3 has been knocked down via siRNA or CRISPR/Cas9, compared to wild-type controls.

Peptide competition assay: Pre-incubate your antibody with excess immunizing peptide before application to your sample. Specific signals should be abolished, while non-specific binding typically persists.

Orthogonal detection methods: Confirm your findings using multiple antibodies targeting different epitopes of ABI3, or employ alternative detection methods like mass spectrometry to verify protein identity.

Application-specific strategies:

• For IHC/IF: Include isotype control antibodies and perform secondary-only controls

• For IP-WB: Confirm specificity by probing immunoprecipitated material with a second ABI3 antibody targeting a different epitope

• For ELISA: Perform titration curves with known positive and negative samples to establish signal-to-noise ratios

Remember that different applications have different specificity thresholds; Western blotting typically provides higher specificity than IHC due to molecular weight information.

How can I use ABI3 antibodies to investigate its relationship with Alzheimer's disease pathology?

ABI3 has been genetically linked to Alzheimer's disease (AD), with emerging evidence suggesting it contributes to microglia-mediated disease progression by regulating microglial morphology . To investigate this relationship:

Co-immunoprecipitation studies: Use ABI3 antibodies for IP (Cell Signaling #23060 at 1:50 dilution) followed by Western blotting to identify protein interaction partners in microglia that may be altered in AD. This approach can reveal novel signaling pathways affected by ABI3 variants.

Comparative immunohistochemistry: Apply ABI3 antibodies (Abcam ab214318 at 1:200) to tissue sections from AD patients versus controls, quantifying differences in expression patterns, cellular localization, and co-localization with pathological markers like amyloid-β plaques or phosphorylated tau.

Time-course analyses: Examine ABI3 expression and localization across disease progression using animal models of AD, correlating changes with behavioral deficits and pathological hallmarks.

Functional assays: After immunophenotyping with ABI3 antibodies, isolate microglia from AD models to assess phagocytic capacity, cytokine production, and morphological dynamics in response to inflammatory stimuli or Aβ exposure.

Variant-specific approaches: Develop or obtain antibodies that specifically recognize AD-associated ABI3 variants to distinguish their expression and localization patterns from wild-type protein.

These approaches can help elucidate how ABI3 contributes to microglial function in the context of neurodegeneration and potentially identify novel therapeutic targets.

What are the methodological considerations for studying ABI3's role in actin polymerization and cell motility?

ABI3 is implicated in regulating cell motility through actin polymerization in structures such as lamellipodia and filopodia . When investigating these functions:

Live-cell imaging: Combine ABI3 immunofluorescence (using Santa Cruz C-7 antibody) with phalloidin staining to visualize co-localization with F-actin in motile structures. Time-lapse microscopy following stimulation with growth factors can reveal dynamic associations.

Subcellular fractionation: Use biochemical approaches to isolate cytoskeletal fractions, then perform Western blotting with ABI3 antibodies to quantify association with the actin cytoskeleton under different experimental conditions.

Proximity ligation assays: Investigate physical interactions between ABI3 and known binding partners (e.g., ABL1, WAVE complex components) at subcellular resolution in intact cells.

Rescue experiments: In ABI3-depleted cells showing motility defects, reintroduce wild-type or mutant forms of ABI3, then use antibodies to confirm expression levels and localization patterns before assessing functional rescue.

Phosphorylation-state specific analysis: Since cytoskeletal regulations often involve phosphorylation cascades, consider using phospho-specific antibodies (if available) or general phospho-detection after ABI3 immunoprecipitation to correlate modifications with functional outcomes.

These approaches enable mechanistic insights into how ABI3 contributes to cytoskeletal dynamics and cellular behaviors beyond simple localization studies.

How do different commercially available ABI3 antibodies compare in sensitivity and specificity across applications?

Based on the available information, here is a comparative analysis of commercially available ABI3 antibodies:

Sensitivity considerations:

• For detecting low expression levels, monoclonal antibodies like Santa Cruz C-7 may provide better signal-to-noise ratios in Western blotting

• For detecting native protein conformations, polyclonal antibodies like Abcam's may capture more epitopes in IHC

Specificity trade-offs:

• Monoclonal antibodies offer higher epitope specificity but may miss splice variants

• Polyclonal antibodies provide broader epitope recognition but potentially more cross-reactivity

Application optimization:

• For multi-application studies, Santa Cruz's antibody offers the broadest validated application range

• For specific applications, consider primary validation data (e.g., Abcam for IHC-P, Cell Signaling for WB/IP)

Researchers should conduct preliminary validation in their specific experimental systems before committing to large-scale studies with any antibody.

How can I integrate ABI3 antibody-based studies with other molecular techniques to comprehensively understand its function?

To develop a comprehensive understanding of ABI3 function, integrate antibody-based techniques with complementary approaches:

Genomic-proteomic integration:

• Correlate ABI3 protein levels detected by Western blotting with mRNA expression from qPCR or RNA-seq

• Use ABI3 antibodies to perform ChIP-seq to identify potential transcriptional regulatory networks

• Compare protein localization (via IF/IHC) with single-cell transcriptomics to understand cell type-specific expression patterns

Structural-functional correlations:

• Combine immunoprecipitation using ABI3 antibodies with mass spectrometry to identify interaction partners

• Use proximity labeling approaches (BioID, APEX) followed by ABI3 immunoblotting to validate spatial associations

• Correlate protein domains recognized by different antibodies with functional outcomes in mutagenesis studies

Temporal dynamics:

• Apply ABI3 antibodies in tissue microarrays spanning developmental timepoints or disease progression

• Use fluorescence recovery after photobleaching (FRAP) with fluorescently tagged antibodies to assess protein mobility

• Perform pulse-chase experiments followed by immunoprecipitation to determine protein half-life and turnover rates

Methodological workflow example:

• Initial characterization using Western blotting and immunofluorescence to establish expression patterns

• Co-immunoprecipitation to identify key interaction partners

• CRISPR/Cas9 modification of endogenous ABI3, followed by antibody validation

• Functional assays (phagocytosis, migration) with quantitative immunofluorescence

• In vivo validation using appropriate animal models and IHC

How can ABI3 antibodies be utilized in studying neuroinflammation and microglial function?

ABI3's genetic association with Alzheimer's disease and its role in regulating microglial morphology position it as a valuable target for neuroinflammation research:

Microglial morphology analysis:

• Use ABI3 immunofluorescence to classify microglial activation states based on morphological features and ABI3 expression levels

• Perform time-lapse imaging following inflammatory stimuli to correlate changes in ABI3 localization with microglial morphological transitions

• Apply automated image analysis algorithms to quantify morphological parameters in ABI3-positive versus ABI3-negative microglia

Functional correlations:

• Combine ABI3 immunolabeling with phagocytosis assays using fluorescent beads or disease-relevant substrates (Aβ, myelin debris)

• Assess cytokine production profiles in microglia sorted based on ABI3 expression levels

• Compare chemotactic responses and process motility in cells with different ABI3 expression patterns

Pathological contexts:

• Analyze ABI3 expression in microglia surrounding amyloid plaques versus those in non-plaque regions

• Compare ABI3 immunoreactivity patterns in various neurodegenerative conditions (AD, PD, ALS, MS) to identify disease-specific signatures

• Examine ABI3 expression changes following therapeutic interventions targeting neuroinflammation

Translational approaches:

• Develop tissue-based assays using patient-derived samples to correlate ABI3 variants with microglial phenotypes

• Use ABI3 antibodies to monitor microglial responses to experimental therapeutics in preclinical models

• Establish ABI3 expression patterns as potential biomarkers for microglial activation states in neuroinflammatory conditions

These applications leverage ABI3 antibodies to gain mechanistic insights into the role of microglia in neurological diseases, potentially identifying new therapeutic targets.

What methodological approaches are recommended for investigating ABI3's interaction with tumor suppression pathways?

ABI3 has been implicated in reducing cell motility in vitro and may inhibit tumor metastasis . To investigate its tumor suppressive functions:

Expression correlation studies:

• Use Western blotting and IHC with ABI3 antibodies to compare expression levels between normal tissues, primary tumors, and metastatic lesions

• Correlate expression patterns with clinical outcomes and established prognostic markers

• Perform tissue microarray analysis across tumor grades to establish potential diagnostic value

Functional pathway analysis:

• Conduct co-immunoprecipitation using ABI3 antibodies followed by proteomic analysis to identify cancer-relevant interaction partners

• Use proximity ligation assays to validate key interactions in situ in tumor versus normal tissues

• Examine phosphorylation status of ABI3 in response to oncogenic signaling using phospho-enrichment followed by immunoblotting

Mechanistic investigations:

• Combine ABI3 overexpression/knockdown with invasion and migration assays, using antibodies to confirm manipulation efficiency

• Perform rescue experiments with wild-type versus mutant ABI3 in cancer cell lines, followed by immunofluorescence to assess localization

• Investigate ABI3's interaction with TARSH (a gene associated with cellular senescence) using dual-immunofluorescence and co-IP approaches

Translational research directions:

• Develop tissue-based assays that can stratify patients based on ABI3 expression patterns

• Screen for compounds that stabilize or upregulate ABI3 expression, using antibodies as readouts

• Assess potential for ABI3-targeting therapies by monitoring protein expression in patient-derived xenograft models

These methodological approaches can help elucidate ABI3's role in tumor suppression pathways and potentially identify novel therapeutic strategies for cancers where ABI3 dysregulation occurs.