GAB1 Antibody

What is the molecular structure of GAB1 and why is it significant for antibody selection?

GAB1 (GRB2-associated-binding protein 1) is a multi-substrate docking protein that functions as a signaling adapter molecule. It contains a pleckstrin homology (PH) domain, multiple tyrosine phosphorylation sites, and proline-rich sequences that mediate protein-protein interactions. When selecting antibodies, researchers should consider which domains need to be targeted:

• The protein has 724 amino acids with a calculated molecular weight of approximately 77-80 kDa, although it typically runs at ~110 kDa on SDS-PAGE due to post-translational modifications

• GAB1 contains at least 16 tyrosine phosphorylation sites, some serving as binding sites for PI3K, Grb2, PLCγ1, Nck, and SHP2

• Phosphorylation on tyrosines 627 and 659 is critical for SHP2 binding and ERK/MAPK pathway activation

This structural complexity necessitates careful antibody selection based on the specific domains or modifications being studied.

What are the primary research applications for GAB1 antibodies?

GAB1 antibodies are utilized in multiple experimental techniques to investigate signaling pathways and protein interactions:

Researchers should optimize antibody dilutions for their specific experimental system. For Western blot applications, GAB1 antibodies have been validated in multiple cell lines including HEK-293, K-562, and C6 cells .

How does GAB1 function in different signaling pathways?

GAB1 serves as an integration hub for multiple signaling pathways:

• Growth factor signaling: Transduces signals from tyrosine kinases, including Met, FGFR1, and EGFR

• Cytokine signaling: Mediates IL-6-induced MAPK pathway activation and inflammatory responses

• PDGF signaling: Essential for oligodendrocyte differentiation and myelination in the central nervous system

• HGF/MET signaling: Critical for angiogenesis and blood vessel formation

For research purposes, antibodies targeting different phosphorylation sites can be used to distinguish pathway-specific GAB1 activation.

What are the optimal conditions for using GAB1 antibodies in Western blotting?

Achieving specific and reproducible results with GAB1 antibodies in Western blotting requires protocol optimization:

• Sample preparation: Use lysis buffers containing phosphatase inhibitors if studying phosphorylated GAB1

• Protein loading: Load 10-20 μg of total cell lysate per lane

• Electrophoresis conditions: Use 7.5-10% gels for optimal separation

• Transfer conditions: For the 110 kDa GAB1 protein, longer transfer times or higher voltage may be necessary

• Blocking: 5% non-fat dry milk in TBST is typically effective

• Primary antibody incubation: 1:1000-1:2000 dilution overnight at 4°C

• Detection: Secondary antibody at 1:10,000, followed by standard detection methods

For optimal GAB1 detection, samples from HEK-293, K-562, or A431 cell lines can serve as positive controls .

How should GAB1 antibodies be used for immunohistochemistry applications?

Successful immunohistochemical detection of GAB1 requires specific tissue preparation techniques:

• Fixation: Both paraffin-embedded and frozen tissue sections are suitable

• Antigen retrieval: For FFPE tissues, use either:

  • TE buffer pH 9.0 (recommended)

  • Citrate buffer pH 6.0 (alternative)

• Blocking: 1% BSA, 0.3% Triton, 10% goat serum for 1 hour at room temperature

• Primary antibody: Dilute 1:50-1:500 and incubate overnight at 4°C

• Secondary antibody: Apply at 1:1000 dilution for 1 hour at room temperature

GAB1 exhibits cytoplasmic, membranous, and nuclear localization patterns. Control tissues with confirmed GAB1 expression include breast, prostate, testis, tonsil, stomach, and transitional cell carcinoma .

What controls should be implemented when using GAB1 antibodies?

Proper experimental controls are essential for valid interpretation of GAB1 antibody results:

• Positive controls:

  • Cell lines: HEK-293, K-562, MCF-7, A431, and NIH-3T3 cells show endogenous GAB1 expression

  • Tissues: Testis, breast, and brain tissues demonstrate reliable GAB1 expression

• Negative controls:

  • Isotype controls: Use appropriate isotype-matched antibodies (e.g., rabbit IgG for rabbit polyclonal antibodies)

  • GAB1 knockdown: Lentiviral shRNA targeting GAB1 (sequence: 5'-GCGATAGATCCAGTTCCTTGG-3') can be used to validate antibody specificity

• Stimulation controls:

  • Treat cells with growth factors or cytokines known to induce GAB1 phosphorylation (e.g., IL-6, IL-3, HGF, EGF) to verify antibody sensitivity to activation state

How can GAB1 antibodies be used to investigate protein-protein interactions?

GAB1 functions as a scaffolding protein, making protein interaction studies particularly valuable:

• Co-immunoprecipitation (Co-IP):

  • Use 1:50 dilution of GAB1 antibody for immunoprecipitation

  • Verify interactions with SHP2, p85 (PI3K), PLCγ, and other binding partners

  • Following IL-6 or IL-3 stimulation, SHP2 and p85 can be detected in GAB1 immunoprecipitates

• Proximity ligation assay (PLA):

  • Allows visualization of protein interactions in situ

  • Combine GAB1 antibody with antibodies against suspected binding partners

  • Particularly useful for transient interactions following receptor activation

• Pull-down validation:

  • After Co-IP with GAB1 antibody, perform reverse Co-IP with antibodies against binding partners

  • This bidirectional validation confirms specific interactions

What strategies are recommended for studying GAB1 phosphorylation dynamics?

GAB1 phosphorylation occurs rapidly following receptor activation and regulates downstream signaling:

• Stimulus-specific phosphorylation:

  • IL-6 and IL-3 induce GAB1 tyrosine phosphorylation within 5 minutes

  • HGF/Met signaling induces distinct phosphorylation patterns compared to VEGF

• Phospho-specific antibodies:

  • Target key phosphorylation sites (Y627, Y659) that mediate SHP2 binding

  • Use phosphatase inhibitors during sample preparation

  • Include both total GAB1 and phospho-GAB1 antibodies in parallel samples

• Temporal analysis:

  • Create time-course experiments (0-60 minutes) following stimulation

  • Compare phosphorylation kinetics across different stimuli to identify pathway-specific patterns

How can GAB1 antibodies be utilized in genetic manipulation studies?

GAB1 antibodies are valuable tools for validating genetic modifications in research models:

• Knockout validation:

  • Complete GAB1 knockout is embryonically lethal, necessitating conditional approaches

  • Tissue-specific knockouts can be generated using Cre-loxP systems

  • GAB1 antibodies confirm absence of protein in conditional knockout tissues

• Domain-specific mutations:

  • GAB1 constructs lacking specific domains (e.g., GAB1ΔPH, MBD domain constructs) can be expressed in cells

  • Compare antibody detection patterns between wild-type and mutant proteins

  • Verify expression and localization of mutant constructs

• Knockdown efficiency assessment:

  • Lentiviral shRNA targeting GAB1 (5'-GCGATAGATCCAGTTCCTTGG-3') reduces expression

  • Use Western blotting with GAB1 antibody to quantify knockdown efficiency

  • Correlate knockdown levels with phenotypic outcomes

What are common challenges when using GAB1 antibodies and how can they be addressed?

Researchers frequently encounter specific technical issues when working with GAB1 antibodies:

• Multiple bands in Western blot:

  • GAB1 has family members (GAB2, GAB3) with similar molecular weights

  • Some antibodies may cross-react with these homologs

  • Solution: Use validated monoclonal antibodies with confirmed specificity

• Weak or absent signal:

  • For concentrated antibodies, centrifuge prior to use to ensure recovery of all product

  • Extend primary antibody incubation time to overnight at 4°C

  • For Western blot, increase protein loading to 20-30 μg

• Background issues in immunohistochemistry:

  • Optimize blocking conditions (try 5% BSA instead of serum)

  • Reduce primary antibody concentration

  • Perform additional washing steps

How do sample preparation methods affect GAB1 antibody performance?

Sample preparation significantly impacts GAB1 detection quality:

• Cell lysis considerations:

  • Use buffers containing phosphatase inhibitors when studying phosphorylated GAB1

  • RIPA buffer is suitable for most applications

  • For co-immunoprecipitation, milder NP-40 or Triton X-100 based buffers preserve protein-protein interactions

• Tissue preparation:

  • For paraffin sections, antigen retrieval with TE buffer (pH 9.0) is recommended

  • For frozen sections, brief fixation in 4% paraformaldehyde (15 minutes) preserves antigenicity

  • GAB1 detection works in both paraffin and frozen tissue preparations

• Storage conditions:

  • Store antibodies at -20°C for long-term stability

  • Aliquot to avoid freeze-thaw cycles

  • Most GAB1 antibodies remain stable for one year after shipment

How can GAB1 antibodies be applied in disease-focused research?

GAB1 has been implicated in various pathological conditions, where antibody-based detection provides valuable insights:

• Cancer research applications:

  • GAB1 overexpression correlates with metastasis in breast cancer

  • Elevated GAB1 indicates poor prognosis in hepatocellular carcinoma and epithelial ovarian cancer

  • GAB1 is overexpressed in adult acute lymphoblastic leukemia and medulloblastomas

  • Use GAB1 antibodies for tissue microarray analysis to correlate expression with patient outcomes

• Neurological disorders:

  • GAB1 is essential for oligodendrocyte development and CNS myelination

  • Analyze GAB1 expression in demyelinating disease models

  • Compare GAB1 levels in normal versus diseased brain tissue sections

• Cardiovascular research:

  • GAB1 plays a critical role in postnatal angiogenesis

  • Evaluate GAB1 expression in HGF-dependent versus VEGF-dependent angiogenesis models

  • Monitor GAB1 phosphorylation following ischemic injury and reperfusion

How can GAB1 antibodies be integrated into multi-parameter analysis techniques?

Contemporary research increasingly employs multiplexed approaches:

• Multi-color immunofluorescence:

  • Combine GAB1 antibody with markers for specific cell types or signaling pathways

  • Use different fluorophore-conjugated secondary antibodies

  • Example panel for brain tissue: GAB1 + Olig2 (oligodendrocyte lineage) + CC1 (mature oligodendrocytes) + PDGFRα (OPC marker)

• Mass cytometry (CyTOF):

  • Metal-conjugated GAB1 antibodies can be integrated into panels with 30+ markers

  • Enables single-cell analysis of GAB1 expression in heterogeneous populations

  • Correlate GAB1 levels with cellular phenotypes and activation states

• Spatial transcriptomics correlation:

  • Compare GAB1 protein expression (by IHC) with spatial transcriptomic data

  • Identify discrepancies between mRNA and protein levels

  • Map GAB1 activation patterns in tissue microenvironments

What considerations are important when designing experiments to study GAB1 in conditional knockout models?

Conditional knockout approaches circumvent embryonic lethality of complete GAB1 deletion:

• Breeding strategies:

  • Gab1f/f mice can be crossed with tissue-specific Cre lines

  • Use Gab1f/+; Cre mice as controls to minimize Cre insertion effects

  • GAB1 antibodies confirm knockout efficiency in target tissues

• Phenotypic analysis:

  • In oligodendrocyte-specific knockout (Gab1f/f; Olig1-Cre), myelination defects occur by P21

  • In endothelial-specific knockout, angiogenesis defects are observed in adulthood

  • Use GAB1 antibodies to verify tissue-specific deletion while confirming expression in non-targeted tissues

• Compensatory mechanisms:

  • Monitor potential upregulation of GAB2 or GAB3 in GAB1-deficient tissues

  • Compare phosphorylation of downstream targets (ERK, AKT) in wild-type versus knockout tissues

  • Investigate alterations in binding partner distribution following GAB1 deletion