GAB1 Antibody, FITC conjugated

What is GAB1 and what are its key functions in cellular signaling pathways?

GAB1 (GRB2-associated binding protein 1) functions as an adapter protein that plays crucial roles in intracellular signaling cascades triggered by activated receptor-type kinases. It serves as a docking platform for multiple signaling molecules, facilitating signal transduction across various pathways. GAB1 is involved in several critical signaling networks including FGFR1 signaling, EGFR signaling, insulin receptor signaling, and the MET/HGF-signaling pathway .

Research has demonstrated that GAB1 regulates numerous cellular processes including proliferation, migration, invasion, and apoptosis. Studies in oral squamous cell carcinoma (OSCC) have revealed that GAB1 promotes malignant progression through activation of the Akt/Cdh1 signaling pathway, highlighting its potential importance in cancer biology .

What are the typical applications for FITC-conjugated GAB1 antibodies in research workflows?

FITC-conjugated GAB1 antibodies are valuable tools in multiple research applications:

The direct FITC conjugation eliminates the need for secondary antibody incubation steps, reducing protocol time and minimizing background from cross-reactivity issues. This makes these antibodies particularly valuable for multiparameter analyses where multiple targets need to be detected simultaneously .

How does GAB1 expression vary across different cell and tissue types?

GAB1 expression exhibits significant variation across different biological systems, which has important implications for experimental design and control selection:

According to current research findings, GAB1 shows elevated expression in several cancer cell lines compared to normal counterparts. For Western blot applications, HEK-293, K-562, and C6 cells have been validated as positive controls for GAB1 expression . In immunohistochemistry applications, mouse brain tissue has been confirmed to express detectable levels of GAB1 .

Studies in oral squamous cell carcinoma have demonstrated that GAB1 is overexpressed in OSCC tissues and multiple OSCC cell lines (including SCC15, SCC25, HN4, and HN6) compared to normal human oral keratinocytes (NHOK) . This differential expression correlates with changes in various molecules associated with proliferation, apoptosis, migration, and invasion, including decreased levels of TIMP2, Bax, and Cdh1, and increased levels of Ki-67, Bcl-2, and cyclin D1 .

What controls should be included when using FITC-conjugated GAB1 antibodies in immunofluorescence experiments?

A robust experimental design for immunofluorescence studies using FITC-conjugated GAB1 antibodies requires multiple controls:

• Antibody specificity controls:

  • GAB1 knockout or knockdown samples (generated using CRISPR-Cas9 or siRNA)

  • Isotype control antibody (FITC-conjugated IgG of same species and isotype)

  • Pre-absorption control with recombinant GAB1 protein

• Technical controls:

  • Unstained samples to assess autofluorescence

  • Single-color controls for spectral overlap correction in multicolor experiments

  • Secondary antibody-only controls for any indirect methods

• Biological reference controls:

  • Positive control samples (cell lines with validated GAB1 expression such as HEK-293, K-562, or C6 cells)

  • Samples with manipulated signaling conditions (e.g., stimulated vs. unstimulated)

  • Pathway inhibitor treatments (e.g., LY294002 for Akt inhibition)

For optimal GAB1 detection in mouse brain tissue, antigen retrieval with TE buffer (pH 9.0) is recommended, though citrate buffer (pH 6.0) may be used as an alternative .

How can researchers effectively study GAB1's role in the Akt/Cdh1 signaling pathway?

Based on recent findings, GAB1 plays a critical role in regulating the Akt/Cdh1 signaling pathway, particularly in cancer progression. An effective experimental approach should include:

• Expression analysis:

  • Quantify GAB1, phospho-Akt, and Cdh1 protein levels using Western blot

  • Use FITC-conjugated GAB1 antibodies for cellular localization studies

  • Compare expression in normal versus cancerous tissues/cells

• Genetic manipulation:

  • Deploy siRNA-mediated knockdown of GAB1 and assess effects on Akt phosphorylation and Cdh1 expression

  • Overexpress GAB1 and evaluate downstream changes in signaling components

  • Perform rescue experiments with mutant GAB1 variants lacking specific domains

• Pharmacological intervention:

  • Use Akt inhibitors (e.g., LY294002) to determine if GAB1-mediated Cdh1 downregulation is reversed

  • Apply specific pathway inhibitors to dissect GAB1's role in different signaling contexts

Research has demonstrated that GAB1 knockdown in SCC15 oral cancer cells decreases phospho-Akt levels while increasing Cdh1 expression. Similarly, Akt inhibition with LY294002 increases Cdh1 expression without affecting GAB1 levels, confirming the sequential nature of the GAB1/Akt/Cdh1 pathway .

What are the optimal sample preparation methods for detecting GAB1 in different experimental applications?

Sample preparation protocols must be optimized based on the specific application and target tissues:

For phosphorylation status studies, it is crucial to include phosphatase inhibitors throughout all preparation steps to preserve the native phosphorylation state of GAB1 and its associated proteins .

How can FITC-conjugated GAB1 antibodies be used to investigate GAB1's role in cancer progression?

FITC-conjugated GAB1 antibodies offer several approaches to study GAB1's role in cancer progression:

• Expression profiling across cancer types:

  • Flow cytometric analysis of GAB1 expression in patient-derived samples

  • Correlation of expression levels with clinical outcomes and disease stages

  • Multiparameter analysis with markers of proliferation (Ki-67) and anti-apoptosis (Bcl-2)

• Functional studies in cancer models:

  • Monitoring GAB1 expression changes following drug treatments

  • Tracking GAB1-expressing cells in invasion and migration assays

  • Correlation between GAB1 expression and therapy resistance

• Mechanistic investigations:

  • Live-cell imaging of GAB1 localization during cancer cell invasion

  • Co-localization studies with receptor tyrosine kinases and downstream effectors

  • FACS-based isolation of GAB1-high vs. GAB1-low populations for transcriptomic analysis

Research has demonstrated that GAB1 is overexpressed in oral squamous cell carcinoma tissues and cell lines compared to normal counterparts. This overexpression correlates with decreased levels of tumor suppressors (TIMP2, Bax, Cdh1) and increased levels of proliferation markers (Ki-67, Bcl-2, cyclin D1) . siRNA-mediated knockdown of GAB1 inhibits proliferation and invasion while promoting apoptosis in OSCC cell lines, suggesting GAB1 as a potential therapeutic target .

What methodological approaches can resolve discrepancies between theoretical and observed molecular weights of GAB1?

Addressing the discrepancy between calculated (80 kDa) and observed (110 kDa) molecular weights of GAB1 requires systematic investigation:

• Protein modification analysis:

  • Phosphorylation mapping using phosphatase treatments and phospho-specific antibodies

  • Glycosylation assessment through treatment with glycosidases

  • Ubiquitination analysis using deubiquitinating enzymes

• Domain-specific detection:

  • Use of antibodies targeting different epitopes within GAB1

  • Comparison between N-terminal and C-terminal targeting antibodies

  • Expression of truncated GAB1 constructs to identify regions contributing to anomalous migration

• Advanced protein characterization:

  • Mass spectrometry analysis to determine actual molecular weight

  • 2D gel electrophoresis to resolve post-translationally modified isoforms

  • Comparative analysis across different sample preparation methods and buffer systems

The observed 110 kDa band for GAB1 in Western blot analysis is consistently reported across multiple studies and antibodies, suggesting this discrepancy is intrinsic to the protein rather than an artifact of specific detection methods . Post-translational modifications, particularly phosphorylation at multiple sites, likely contribute to this reduced electrophoretic mobility.

How can researchers effectively study the interaction between GAB1 and other signaling proteins using fluorescence-based methods?

Fluorescence-based methods offer powerful approaches to study GAB1 interactions with signaling partners:

• Co-immunoprecipitation coupled with fluorescence detection:

  • Immunoprecipitate GAB1 and probe for interacting partners

  • Use FITC-conjugated antibodies for detection in Western blots

  • Perform reciprocal IPs to confirm interactions

• Proximity-based assays:

  • Proximity Ligation Assay (PLA) to visualize interactions within 40nm

  • Fluorescence Resonance Energy Transfer (FRET) for detecting direct interactions

  • Bimolecular Fluorescence Complementation (BiFC) for visualizing protein complexes

• Live-cell interaction dynamics:

  • Fluorescence recovery after photobleaching (FRAP) to assess binding dynamics

  • Fluorescence correlation spectroscopy (FCS) to measure diffusion rates of complexes

  • Single-molecule tracking of GAB1 interactions

Research has established that GAB1 interacts with multiple signaling components including GRB2, PI3K, and SHP2. In the context of cancer progression, the interaction between GAB1 and components of the Akt pathway is particularly significant, as it leads to downstream regulation of Cdh1 and affects cellular proliferation and invasion . The GAB1/Akt/Cdh1 signaling axis has been confirmed through combined approaches of genetic manipulation and pharmacological inhibition .

What are common issues when using FITC-conjugated GAB1 antibodies, and how can they be addressed?

Researchers may encounter several technical challenges when working with FITC-conjugated GAB1 antibodies:

• High background fluorescence:

  • Cause: Insufficient blocking, non-specific binding

  • Solution: Increase blocking time/concentration, add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

  • Alternative approach: Use image analysis software to subtract background

• Photobleaching:

  • Cause: FITC susceptibility to photobleaching under extended exposure

  • Solution: Add anti-fade reagents to mounting media, minimize exposure during imaging

  • Alternative approach: Consider photostable alternatives like Alexa Fluor 488

• Weak signal intensity:

  • Cause: Low GAB1 expression, epitope masking, or suboptimal fixation

  • Solution: Optimize antibody concentration (1:50-1:500 range for IHC applications)

  • Alternative approach: Try different antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Unexpected cellular localization:

  • Cause: Fixation artifacts, cross-reactivity, or cell-state dependent localization

  • Solution: Compare multiple fixation methods, validate with GAB1 knockdown controls

  • Alternative approach: Use alternative GAB1 antibody clones targeting different epitopes

• Inconsistent results across samples:

  • Cause: Variable GAB1 expression levels or post-translational modifications

  • Solution: Include standardized positive controls (HEK-293, K-562, C6 cells)

  • Alternative approach: Normalize detection using total protein stains or housekeeping proteins

How can researchers distinguish between specific and non-specific binding when using GAB1 antibodies?

Validating antibody specificity is crucial for accurate interpretation of results:

• Genetic approach validation:

  • Use GAB1 knockout or knockdown cells as negative controls

  • Express exogenous GAB1 in low-expressing cells as positive controls

  • Compare staining patterns between wildtype and GAB1-manipulated samples

• Biochemical validation:

  • Perform peptide competition assays by pre-incubating antibody with immunizing peptide

  • Compare multiple GAB1 antibodies targeting different epitopes

  • Confirm binding to recombinant GAB1 protein of known concentration

• Technical validation:

  • Include isotype controls to assess non-specific binding

  • Use appropriate blocking agents to minimize background

  • Perform titration experiments to determine optimal antibody concentration

• Analytical validation:

  • Compare patterns across multiple detection methods (IF, WB, IHC)

  • Confirm expected molecular weight in Western blot (observed at 110 kDa)

  • Verify subcellular localization pattern consistent with GAB1's known biology

In Western blot applications, true GAB1 signal should appear at approximately 110 kDa despite a calculated molecular weight of 80 kDa, which is attributed to post-translational modifications . This characteristic migration pattern can serve as an important specificity control.

What steps should be taken to optimize signal-to-noise ratio in immunofluorescence experiments with FITC-conjugated GAB1 antibodies?

Maximizing signal-to-noise ratio requires systematic optimization:

• Sample preparation optimization:

  • Test multiple fixation protocols (4% PFA, methanol, or combination)

  • Compare different permeabilization agents (Triton X-100, saponin, digitonin)

  • Optimize antigen retrieval methods (TE buffer pH 9.0 recommended for GAB1)

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time (1-2 hours at room temperature)

  • Add 0.1-0.3% detergent to blocking buffer to reduce hydrophobic interactions

• Antibody incubation optimization:

  • Perform titration to determine optimal concentration

  • Test different incubation temperatures and durations

  • Consider using antibody diluents with background-reducing components

• Washing optimization:

  • Increase number and duration of washes

  • Use gentle agitation during washing steps

  • Consider detergent concentration in wash buffers

• Microscopy settings optimization:

  • Adjust exposure settings to avoid saturation

  • Implement background subtraction in image analysis

  • Use appropriate filters to minimize spectral bleed-through

For positive controls, mouse brain tissue has been validated for GAB1 detection in immunohistochemistry applications, and the recommended dilution range for immunohistochemistry is 1:50-1:500 .

How can FITC-conjugated GAB1 antibodies be integrated with other methodologies to study the GAB1/Akt/Cdh1 pathway in cancer?

An integrated approach to studying the GAB1/Akt/Cdh1 pathway combines multiple methodologies:

• Multiparameter analysis:

  • Simultaneous detection of GAB1, phospho-Akt, and Cdh1 using differently labeled antibodies

  • Correlation of GAB1 expression with markers of proliferation (Ki-67) and anti-apoptosis (Bcl-2)

  • Integration of protein and mRNA expression data

• Combinatorial intervention studies:

  • Genetic manipulation (siRNA, CRISPR) combined with pharmacological inhibition

  • Dual targeting of GAB1 and Akt to assess synergistic effects

  • Rescue experiments with constitutively active Akt or GAB1 mutants

• Translational research approach:

  • Patient-derived xenograft models analyzed with FITC-GAB1 antibodies

  • Correlation between GAB1 expression and treatment response

  • Development of GAB1-targeted therapies based on pathway understanding

Research has established that GAB1 knockdown increases Cdh1 expression while decreasing phospho-Akt levels. Similarly, pharmacological inhibition of Akt with LY294002 increases Cdh1 expression without affecting GAB1 levels, confirming the sequential GAB1→Akt→Cdh1 relationship . These findings suggest that dual targeting of this pathway may have therapeutic potential in GAB1-overexpressing cancers.

What methodological approaches can determine if GAB1 phosphorylation status correlates with its functional activity?

Investigating the relationship between GAB1 phosphorylation and function requires specialized approaches:

• Phosphorylation site mapping:

  • Use phospho-specific antibodies targeting different GAB1 residues

  • Employ mass spectrometry to identify all phosphorylation sites

  • Create phosphomimetic and phospho-dead mutants for functional studies

• Temporal dynamics analysis:

  • Time-course experiments following receptor activation

  • Correlation between phosphorylation timing and downstream signaling events

  • Real-time monitoring of phosphorylation using biosensors

• Spatial phosphorylation patterns:

  • Use FITC-conjugated total GAB1 antibody with other fluorophore-conjugated phospho-specific antibodies

  • Analyze subcellular distribution of phosphorylated vs. total GAB1

  • Three-dimensional reconstruction of phospho-GAB1 distribution

• Functional correlation:

  • Compare phosphorylation status with binding to downstream effectors

  • Correlate phosphorylation patterns with cellular outcomes (proliferation, migration)

  • Use phosphatase inhibitors to preserve phosphorylation status during sample preparation

Research suggests that GAB1 phosphorylation status significantly impacts its ability to activate the Akt pathway, which subsequently regulates Cdh1 expression and influences cellular processes including proliferation, invasion, and apoptosis .

How can researchers design experiments to investigate whether GAB1 could serve as a therapeutic target in cancer?

Evaluating GAB1 as a potential therapeutic target requires a systematic experimental approach:

• Target validation studies:

  • Correlate GAB1 expression with patient survival across cancer types

  • Perform genetic depletion studies in multiple cancer cell lines

  • Evaluate GAB1 knockdown effects in patient-derived xenograft models

• Mechanism-based intervention:

  • Design inhibitors targeting GAB1 protein-protein interactions

  • Develop strategies to block GAB1 phosphorylation

  • Identify synthetic lethal interactions with GAB1 overexpression

• Combination therapy assessment:

  • Test GAB1 inhibition with standard chemotherapeutics

  • Evaluate synergy with targeted therapies (e.g., Akt inhibitors)

  • Determine if GAB1 targeting can overcome therapy resistance

• Biomarker development:

  • Use FITC-conjugated GAB1 antibodies for patient stratification

  • Correlate GAB1 expression/phosphorylation with treatment response

  • Develop companion diagnostics for GAB1-targeted therapies

Research in oral squamous cell carcinoma has demonstrated that GAB1 silencing inhibits proliferation and invasion while promoting apoptosis in cancer cells . The mechanism involves disruption of the Akt/Cdh1 signaling pathway, suggesting that targeting GAB1 could effectively impair multiple cancer-promoting processes simultaneously. These findings indicate that GAB1 may represent a promising therapeutic target, particularly in cancers with GAB1 overexpression .