GAB2 Antibody, FITC conjugated

What is GAB2 and what are its primary biological functions?

GAB2 is an adapter protein that acts downstream of several membrane receptors including cytokine, antigen, hormone, cell matrix, and growth factor receptors to regulate multiple signaling pathways. It plays crucial roles in:

• Regulating osteoclast differentiation by mediating the TNFRSF11A/RANK signaling

• Facilitating mast cells activation and degranulation through PI-3-kinase regulation during allergic responses

• Controlling cell proliferation mechanisms across multiple cell types

• Maintaining and regulating hematopoiesis, particularly in hematopoietic stem cells

GAB2 is tyrosine phosphorylated by several early acting cytokine receptors such as Flt3, c-Kit, interleukin (IL)-3R, and c-Mpl, and contains binding sites for SH2 and SH3 domains that promote binding to signaling molecules. Through these interactions, GAB2 activates the phosphatidylinositol-3′-kinase (PI3-K) and the mitogen activated protein kinase (MAPK) pathways, regulating cell survival, proliferation, cytoskeleton reorganization, and adhesion/migration functions .

What are the recommended applications for GAB2 antibody, FITC conjugated?

The FITC-conjugated GAB2 antibody is recommended primarily for immunofluorescence techniques. Specifically:

• Immunofluorescence on paraffin-embedded tissue sections (IF(IHC-P)) at dilutions of 1:50-200

• Flow cytometry analysis when combined with appropriate protocols

• ELISA applications, depending on the specific antibody formulation

The antibody demonstrates reactivity against human, mouse, and rat GAB2 proteins, making it versatile for cross-species research applications .

What are the optimal storage conditions for maintaining antibody activity?

For maximum stability and retention of activity:

• Store the antibody at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles as these significantly reduce antibody performance

• The antibody is typically provided in a liquid form containing preservatives (e.g., 0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4)

It is advisable to prepare small aliquots for single-use applications if the antibody will be accessed frequently to prevent degradation from multiple freeze-thaw cycles.

How does the fluorescein isothiocyanate (FITC) conjugation affect antibody applications?

FITC conjugation provides direct fluorescence detection capabilities with the following characteristics:

• FITC has an excitation maximum at approximately 495 nm and emission maximum at 519 nm

• The conjugation eliminates the need for secondary antibodies in immunofluorescence applications

• This reduces background signal and potential cross-reactivity issues in multi-labeling experiments

• The direct conjugation enables more streamlined protocols for flow cytometry and microscopy

• FITC conjugation may slightly reduce antibody affinity compared to unconjugated versions, sometimes necessitating higher concentrations than unconjugated counterparts

How does GAB2 coordinate with STAT5 in hematopoietic stem cell regulation?

GAB2 and STAT5 exhibit a complex relationship in hematopoietic stem cell (HSC) function:

• Both proteins function in non-redundant manners to control HSC survival and self-renewal

• GAB2 may act upstream or downstream of STAT5, a major positive regulator of HSC function

• While STAT5 deficiency leads to competitive multilineage HSC repopulating defects (averaging 36.7±6.4% of wild-type), GAB2 deficiency results in similar but less severe defects (17.5±3.5% of wild-type)

• Surprisingly, combined deficiency in GAB2 and STAT5 (Gab2−/−STAT5ab+/null) leads to synergistic defects with only 9.5±1.7% of wild-type engraftment

• This demonstrates important synergy between STAT5 and GAB2 in HSC maintenance and self-renewal

The relationship between these pathways appears bidirectional - GAB2 can influence STAT5 activation, while STAT5 can also affect GAB2-mediated signaling through complex feedback mechanisms.

What methodological approaches can distinguish between GAB2's roles in different signaling pathways?

To differentiate GAB2's functions in various signaling contexts:

• Mutational analysis: Using mutant forms of GAB2 with specific binding site alterations:

  • GAB2 mutants lacking SHP2 binding sites inhibit macrophage differentiation and MAPK activation

  • These mutants result in increased growth potential in response to M-CSF

  • This approach can isolate the importance of specific protein-protein interactions

• Pathway inhibition studies:

  • Selective inhibition of PI3K versus MAPK pathways can help determine which downstream effector is critical for specific GAB2-mediated functions

  • Combined with GAB2 antibody staining, this can reveal pathway-specific alterations in GAB2 phosphorylation and localization

• Cell-type specific analyses:

  • Compare GAB2 signaling in mast cells (allergic response) versus osteoclasts (differentiation)

  • Use the FITC-conjugated antibody to track GAB2 localization and abundance across different cellular contexts

How can cross-talk between JAK/STAT5 and PI3-K pathways be effectively studied using GAB2 antibodies?

The complex interplay between these pathways can be investigated through:

• Co-immunoprecipitation studies:

  • Using GAB2 antibodies to pull down protein complexes

  • Probing for associated proteins like STAT5, PI3K components, and other signaling molecules

  • Analyzing how these associations change with different cytokine stimulations

• Time-course analysis:

  • FITC-conjugated GAB2 antibodies can track the temporal dynamics of GAB2 localization

  • This can be combined with phospho-specific antibodies against STAT5 and PI3K pathway components

  • Research has shown that in response to IL-2, a delayed cycloheximide-sensitive mechanism for coordinated cyclin D2 expression involves both PI3-K and STAT5 activation

• Knockdown/overexpression studies:

  • Silencing or overexpressing GAB2 while monitoring effects on STAT5 and PI3K activities

  • STAT5 knockdown has been shown to impair IL-2 induced Shc-mediated stimulation of Akt activation, suggesting GAB2 might function downstream of STAT5

What are the optimal protocols for using FITC-conjugated GAB2 antibody in immunofluorescence studies?

For optimal immunofluorescence results with paraffin-embedded tissues:

• Sample preparation:

  • Fix tissues in 10% neutral buffered formalin

  • Process and embed in paraffin following standard protocols

  • Section tissues at 4-6 μm thickness

• Antigen retrieval:

  • Deparaffinize and rehydrate sections

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Allow sections to cool to room temperature

• Blocking and antibody incubation:

  • Block with 5-10% normal serum in PBS for 1 hour at room temperature

  • Dilute the FITC-conjugated GAB2 antibody at 1:50-1:200 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber protected from light

• Counterstaining and mounting:

  • Wash sections thoroughly with PBS

  • Counterstain nuclei with DAPI

  • Mount with an anti-fade mounting medium specifically designed for fluorescence

• Controls:

  • Include a negative control by omitting the primary antibody

  • Include positive control tissues known to express GAB2

  • Consider using tissues from GAB2 knockout models if available for specificity validation

How can flow cytometry protocols be optimized for GAB2 detection?

For effective flow cytometry using FITC-conjugated GAB2 antibody:

• Cell preparation:

  • Harvest cells of interest (e.g., hematopoietic cells, mast cells)

  • Wash in cold PBS containing 1% BSA

  • Fix cells with 4% paraformaldehyde for 15 minutes if intracellular staining is required

• Permeabilization for intracellular staining:

  • Since GAB2 is predominantly intracellular, permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer

  • Incubate for 15 minutes at room temperature

• Antibody staining:

  • Block with 5% normal serum from the same species as secondary antibody (if using)

  • Incubate with FITC-conjugated GAB2 antibody at manufacturer's recommended dilution

  • For similar applications, protocols have used polyclonal antibodies followed by FITC-conjugated secondary antibodies for flow cytometric analysis

• Analysis parameters:

  • Set appropriate compensation when using multiple fluorochromes

  • Analyze FITC signal in FL1 channel (typically ~530/30 nm bandpass filter)

  • Include unstained controls and isotype controls for proper gating

What strategies can minimize photobleaching of FITC during extended imaging sessions?

FITC is susceptible to photobleaching, which can be mitigated through:

• Anti-fade reagents:

  • Use mounting media containing anti-fade compounds (e.g., DABCO, PPD)

  • Commercial anti-fade solutions specifically designed for FITC can extend fluorescence stability

• Imaging techniques:

  • Reduce exposure time and light intensity during acquisition

  • Use neutral density filters to attenuate excitation light

  • Consider confocal microscopy with line scanning instead of point scanning

  • Implement deconvolution algorithms to achieve high signal-to-noise with lower excitation intensities

• Sample preparation:

  • Seal slides with nail polish to prevent oxidation

  • Store slides at 4°C in the dark when not imaging

  • Consider using glycerol-based mounting media with anti-fade properties

• Alternative approaches:

  • For critical samples, consider acquiring images of multiple fields immediately

  • For long time-course studies, consider more photostable fluorophores or alternative detection methods

How can researchers differentiate between specific and non-specific binding when using GAB2 antibody?

To ensure specificity and minimize false positives:

• Control experiments:

  • Use tissues/cells from GAB2 knockout models as negative controls

  • Pre-absorb the antibody with recombinant GAB2 protein before staining to validate specificity

  • Compare staining patterns with published literature and established GAB2 localization patterns

• Validation approaches:

  • Verify results using a second GAB2 antibody recognizing a different epitope

  • Confirm findings with complementary techniques (e.g., western blot, RT-PCR)

  • Use siRNA knockdown of GAB2 to demonstrate reduction in staining intensity

• Technical considerations:

  • Optimize blocking conditions to reduce background

  • Ensure proper permeabilization for intracellular targets

  • Compare staining patterns at multiple antibody dilutions to identify optimal signal-to-noise ratio

What are the critical factors in experimental design when studying GAB2's role in hematopoietic stem cell function?

When investigating GAB2 in HSC biology, consider:

• Model systems selection:

  • Utilize genetic models (Gab2−/−, STAT5ab+/null, Gab2−/−STAT5ab+/null)

  • Choose appropriate cell populations (KLS cells, long-term HSCs)

  • Select relevant cytokine conditions that activate GAB2-dependent pathways

• Functional readouts:

  • Competitive repopulation assays to assess HSC function (as demonstrated in studies showing Gab2−/− BM had competitive multilineage HSC repopulating defects averaging 17.5±3.5% of wild-type)

  • Colony-forming assays to evaluate progenitor activity

  • Cytokine stimulation assays to measure proliferation and differentiation responses

• Mechanistic analyses:

  • Investigate phosphorylation status of GAB2 and downstream effectors

  • Examine interactions between GAB2 and key binding partners (SHP2, PI3K)

  • Analyze alterations in gene expression profiles following manipulation of GAB2 expression or function

• Temporal considerations:

  • Assess both immediate signaling events and long-term functional outcomes

  • Design experiments that capture both steady-state hematopoiesis and stress hematopoiesis

  • Longitudinal studies to track HSC behavior over time after perturbation of GAB2 activity

How should researchers approach contradictory results between GAB2 antibody staining and functional outcomes?

When faced with discrepancies:

• Technical validation:

  • Verify antibody lot consistency and specificity

  • Ensure proper controls are included

  • Validate findings with alternative detection methods

• Biological complexity considerations:

  • GAB2 functions differently across cell types and contexts

  • Compensatory mechanisms may exist in chronic models (knockout) versus acute models (inhibition)

  • Cross-talk between pathways may mask or exaggerate phenotypes

• Experimental design refinement:

  • Implement dose-response studies

  • Conduct time-course experiments to capture temporal dynamics

  • Utilize more specific tools (domain-specific mutants, as shown in studies where GAB2 mutants lacking SHP2 binding sites affected MAPK activity differently)

• Integrated data analysis:

  • Combine protein-level data (antibody staining) with functional readouts

  • Correlate findings with gene expression or epigenetic data

  • Consider systems biology approaches to model complex GAB2 signaling networks

What emerging applications exist for studying GAB2 in non-hematopoietic tissues?

While GAB2 has been extensively studied in hematopoietic cells, its functions extend to:

• Neurological contexts:

  • Both GAB2 and GABRG2 antibodies are used in neuroscience research

  • Cross-talk between JAK/STAT5 and PI3-K pathways has been described in neural cells and is important for pro-survival signaling

  • Potential roles in neuroinflammation and neural stem cell function remain to be fully explored

• Cancer biology:

  • GAB2 activates pathways critical for cell survival and proliferation

  • The regulation of cell proliferation by GAB2 suggests potential roles in oncogenesis

  • FITC-conjugated antibodies could be valuable for tracking GAB2 expression and localization in cancer models

• Immunological disorders:

  • Given GAB2's role in mast cell activation and allergic responses, further investigation in autoimmune and inflammatory conditions is warranted

  • The antibody could be utilized to track GAB2 dynamics during immune responses in various tissues

How might novel microscopy techniques enhance the utility of FITC-conjugated GAB2 antibodies?

Advanced imaging approaches offer new possibilities:

• Super-resolution microscopy:

  • Techniques like STORM, PALM, or STED can overcome the diffraction limit

  • These approaches could reveal GAB2 molecular clustering and nanoscale organization not visible with conventional microscopy

  • May uncover previously unknown spatial relationships between GAB2 and binding partners

• Live-cell imaging adaptations:

  • While FITC-conjugated antibodies are typically used in fixed cells, membrane-permeable derivatives could enable live dynamics studies

  • Alternatively, correlative approaches combining live imaging with fixed cell GAB2 immunofluorescence

• Multiplexed imaging:

  • Combining FITC-GAB2 antibody with spectral unmixing techniques

  • Sequential staining approaches to visualize multiple signaling components simultaneously

  • Mass cytometry adaptations to analyze dozens of parameters alongside GAB2 expression