TSC2 Antibody, FITC conjugated

What is TSC2 and what is its biological significance?

TSC2, also known as Tuberin, is a tumor suppressor protein that forms a functional complex with TSC1 (Hamartin). This complex plays an essential role in cellular energy response pathways by negatively regulating mTORC1 signaling. It inhibits the nutrient-mediated or growth factor-stimulated phosphorylation of S6K1 and EIF4EBP1 . The protein has a canonical length of 1807 amino acid residues and a molecular weight of approximately 200.6 kDa . TSC2 specifically stimulates the intrinsic GTPase activity of RAP1A and RAB5, suggesting a mechanism for its role in regulating cellular growth . Mutations in tuberin can lead to constitutive activation of RAP1A in tumors, highlighting its importance in cancer research .

What are the available forms of TSC2 antibodies and their relative advantages?

TSC2 antibodies are available in multiple formats with different characteristics:

The selection should be based on the specific research application, with consideration of factors such as detection method, species reactivity, and epitope of interest .

What are the optimal protocols for using FITC-conjugated TSC2 antibodies in immunofluorescence?

For optimal results with FITC-conjugated TSC2 antibodies in immunofluorescence applications:

• Sample preparation:

  • For cells: Fix with 4% paraformaldehyde (15 minutes), permeabilize with 0.1% Triton X-100 (10 minutes)

  • For tissues: Use freshly frozen or properly fixed paraffin-embedded sections with appropriate antigen retrieval

• Blocking: Block with 5% normal serum from the same species as the secondary antibody (if using one) or BSA for 1 hour at room temperature

• Primary antibody incubation: Apply FITC-conjugated TSC2 antibody at 1:50-1:200 dilution . Incubate overnight at 4°C in a humidified chamber protected from light

• Washing: Wash 3x with PBS containing 0.05% Tween-20

• Counterstaining: Apply nuclear counterstain (e.g., DAPI) if desired

• Mounting: Mount with anti-fade mounting medium

• Visualization: Observe under fluorescence microscope using appropriate filter sets for FITC (excitation ~495nm, emission ~519nm)

Critical considerations include protecting the FITC-conjugated antibody from light throughout the procedure to prevent photobleaching and optimizing the antibody concentration through titration experiments for your specific sample type.

How can I validate the specificity of a TSC2 antibody in my experimental system?

Validating antibody specificity is crucial for reliable results. For TSC2 antibodies, employ the following validation approaches:

• Positive and negative controls:

  • Use cell lines known to express TSC2 (e.g., A549, MCF-7, PC-3, HSC-T6, NIH/3T3, HeLa, U2OS, LNCaP, HEK-293, Jurkat, K-562)

  • Include TSC2-knockout or siRNA-treated samples as negative controls

• Multiple detection methods: Confirm findings using multiple techniques (IF, WB, IHC) to ensure consistent detection

• Peptide competition assay: Pre-incubate antibody with immunizing peptide before application to demonstrate binding specificity

• Molecular weight verification: In Western blot, confirm that the detected band appears at the expected molecular weight (~200 kDa for TSC2)

• Cross-reactivity assessment: Test the antibody against related proteins in the TSC family to ensure specificity

• Phospho-specific validation: For phospho-specific antibodies, treat samples with phosphatase to confirm specificity to the phosphorylated form

What dilution ratios should be used for FITC-conjugated TSC2 antibodies in different applications?

The optimal dilution depends on the specific application and sample type. Based on available data:

It is always advisable to perform a dilution series during initial optimization to determine the optimal concentration for your specific sample and experimental conditions. The signal-to-noise ratio should be evaluated at each dilution to determine the optimal working concentration.

How can I address high background issues when using FITC-conjugated TSC2 antibodies?

High background is a common challenge with fluorescently-labeled antibodies. To reduce background with FITC-conjugated TSC2 antibodies:

• Optimize blocking:

  • Increase blocking time to 2 hours

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

  • Use 0.1-0.3% Triton X-100 in blocking buffer to reduce non-specific membrane binding

• Antibody dilution optimization:

  • Increase dilution factor (use more dilute antibody)

  • Titrate to determine minimal concentration giving specific signal

• Washing improvements:

  • Increase number of washes (5-6 times instead of 3)

  • Extend washing time (10-15 minutes per wash)

  • Add 0.05-0.1% Tween-20 to wash buffer

• Autofluorescence reduction:

  • For tissue sections: Treat with 0.1% Sudan Black B in 70% ethanol for 20 minutes

  • For cells: Short incubation with 0.1% sodium borohydride in PBS

• Fixation optimization:

  • Test different fixatives (PFA, methanol, acetone)

  • Reduce fixation time if overfixation is causing high background

• Sample-specific considerations:

  • For tissues with high endogenous biotin: Use biotin-blocking steps

  • For tissues with high autofluorescence: Consider spectral unmixing during imaging

What are the potential pitfalls in data interpretation when studying TSC2 phosphorylation states?

When analyzing TSC2 phosphorylation with phospho-specific antibodies, consider these interpretation challenges:

How can I quantify the colocalization of TSC2 with other proteins using FITC-conjugated antibodies?

Quantitative colocalization analysis requires careful experimental design and analytical methods:

• Experimental considerations:

  • Use FITC-conjugated TSC2 antibody with spectrally distinct fluorophores for other targets

  • Include appropriate controls (single-stained samples, negative controls)

  • Optimize image acquisition to avoid bleed-through and photobleaching

  • Acquire images at optimal resolution (Nyquist sampling)

• Quantitative colocalization metrics:

  • Pearson's correlation coefficient (PCC): Measures linear correlation (-1 to +1)

  • Manders' overlap coefficient (MOC): Measures percentage overlap (0 to 1)

  • Colocalization rate: Percentage of colocalized pixels relative to total positive pixels

• Analysis workflow:

  • Set appropriate thresholds to exclude background

  • Analyze in 3D when possible to capture complete spatial relationships

  • Use regions of interest (ROIs) to focus on relevant subcellular compartments

  • Apply consistent analysis parameters across all experimental conditions

• Software tools:

  • ImageJ/FIJI with Coloc2 or JACoP plugins

  • Commercial platforms (Imaris, Volocity, ZEN)

  • Custom analysis in Python or MATLAB for complex cases

• Biological validation:

  • Complement imaging with biochemical approaches (co-IP)

  • Disrupt interaction with specific treatments to confirm specificity

  • Use super-resolution techniques for detailed interaction studies

How can I perform multiplexed imaging with FITC-conjugated TSC2 antibodies in TSC pathology studies?

Multiplexed imaging allows simultaneous visualization of multiple targets:

• Compatible fluorophore selection:

  • Pair FITC-conjugated TSC2 antibody (green) with spectrally distinct fluorophores:

    • Alexa Fluor 555/568/594 (red)

    • Alexa Fluor 647/Cy5 (far-red)

    • DAPI/Hoechst (blue, nuclear)

• Sequential staining protocol:

  • For multiple antibodies from the same species: Use tyramide signal amplification (TSA)

  • Apply antibodies sequentially with microwave treatment between rounds to strip previous antibodies

  • Use directly conjugated primary antibodies to avoid species cross-reactivity

• Advanced microscopy techniques:

  • Confocal microscopy for improved optical sectioning

  • Spectral imaging to separate overlapping fluorophores

  • Super-resolution techniques (STED, STORM, SIM) for subcellular localization

• Analysis strategies:

  • Quantify marker coexpression in specific cell populations

  • Measure spatial relationships between TSC2 and other proteins (like TSC1, Rheb, mTOR)

  • Perform neighbor analysis to understand cellular interactions in TSC lesions

• Biological insights in TSC pathology:

  • Correlate TSC2 expression/localization with mTORC1 activation markers

  • Examine cell-type specific alterations in TSC2-deficient lesions

  • Study treatment response by monitoring TSC2-related signaling changes

What are the considerations for studying TSC2 phosphorylation dynamics in live cells?

Live-cell imaging of TSC2 phosphorylation requires specialized approaches:

• Reporter system development:

  • FRET-based biosensors for specific TSC2 phosphorylation sites

  • Split-GFP complementation systems to detect phospho-dependent protein interactions

  • Phospho-binding domain fused to fluorescent proteins

• Advanced microscopy requirements:

  • Spinning disk confocal for fast, low phototoxicity imaging

  • TIRF microscopy for membrane-proximal events

  • Temperature, CO2, and humidity control for physiological conditions

• Stimulation strategies:

  • Microfluidic devices for precise temporal control of stimuli

  • Optogenetic tools for spatially restricted pathway activation

  • Synchronized cell populations for studying cell-cycle dependent effects

• Analysis challenges:

  • Photobleaching correction

  • Cell movement tracking and compensation

  • Signal-to-noise optimization at low light levels

  • Quantification of rapid, subtle changes in phosphorylation

• Biological questions addressable:

  • Temporal relationship between growth factor stimulation and TSC2 phosphorylation

  • Subcellular heterogeneity in phosphorylation responses

  • Oscillatory behaviors in TSC2/mTOR signaling

  • Single-cell variability in response kinetics

How can TSC2 antibodies be integrated into multi-omic approaches for comprehensive pathway analysis?

Integration of TSC2 antibody-based techniques with other omic approaches enhances mechanistic insights:

• Antibody-based sorting for downstream analysis:

  • FACS isolation of TSC2-positive/negative populations for:

    • Transcriptomic analysis (RNA-seq)

    • Proteomic profiling (mass spectrometry)

    • Epigenomic characterization (ATAC-seq, ChIP-seq)

• Spatial multi-omics integration:

  • IF with TSC2 antibodies followed by laser capture microdissection

  • Spatial transcriptomics with protein co-detection

  • CODEX or imaging mass cytometry for highly multiplexed protein analysis

• Pathway reconstruction approaches:

  • Phosphoproteomic analysis to map TSC2-dependent signaling networks

  • Correlation of TSC2 localization with metabolomic profiles

  • Integrated network analysis combining proteomics and transcriptomics data

• Single-cell multi-modal analysis:

  • CITE-seq combining surface protein and transcriptome analysis

  • Single-cell western blot for TSC2 phospho-form analysis

  • Imaging-based single-cell proteomics with TSC2 antibodies

• Data integration strategies:

  • Computational methods for multi-omic data correlation

  • Machine learning approaches to identify patterns across datasets

  • Pathway enrichment analysis incorporating TSC2 interaction networks

What are the latest approaches for studying TSC2 complex formation using advanced microscopy techniques?

Advanced microscopy offers powerful tools for studying TSC2 protein interactions:

• Super-resolution microscopy applications:

  • STORM/PALM for nanoscale localization precision (~20nm)

  • STED microscopy for live-cell super-resolution imaging

  • SIM for improved resolution with standard fluorophores

  • Expansion microscopy for physical sample magnification

• Proximity detection methods:

  • Proximity Ligation Assay (PLA) to visualize TSC1-TSC2 interactions

  • FRET/FLIM to measure direct protein interactions

  • BiFC (Bimolecular Fluorescence Complementation) for stable interaction visualization

• Single-molecule tracking:

  • SPT (Single Particle Tracking) of labeled TSC2 molecules

  • sptPALM for tracking TSC2 complex dynamics

  • FCS (Fluorescence Correlation Spectroscopy) for diffusion behavior analysis

• Quantitative analysis approaches:

  • Particle analysis for TSC2 complex size distribution

  • Diffusion coefficient mapping for membrane/cytoplasmic dynamics

  • Cluster analysis to identify interaction hotspots

• Sample preparation considerations:

  • Membrane sheet preparation for improved access to membrane-associated complexes

  • Optimized fixation protocols to preserve native protein interactions

  • Correlative light-electron microscopy for ultrastructural context

How should I design experiments to study mTORC1 inhibition using TSC2 antibodies?

A comprehensive experimental design for studying mTORC1 inhibition with TSC2 antibodies should include:

• Model system selection:

  • Cell lines: HEK-293, MCF-7, or A549 cells have verified TSC2 expression

  • TSC2-knockout models: CRISPR/Cas9-generated TSC2-/- cells

  • Patient-derived samples: TSC-associated hamartomas vs. normal tissue

• Stimulus and inhibitor panel:

  • Growth factors: Insulin, EGF, IGF-1

  • Nutrients: Amino acids (particularly leucine)

  • Energy stress: Glucose deprivation, 2-DG

  • mTOR inhibitors: Rapamycin, Torin1, Rapalogs

• Readout selection:

  • Direct TSC2 readouts: Total protein levels, phosphorylation state (Tyr1571, Ser1418, Thr1462)

  • Downstream markers: p-S6K (T389), p-4EBP1, p-S6

  • Cellular outcomes: Proliferation, cell size, autophagy markers

• Time-course analysis:

  • Acute responses: 5, 15, 30, 60 minutes

  • Sustained effects: 3, 6, 24, 48 hours

  • Recovery dynamics: Washout experiments

• Technical approaches:

  • Protein analysis: Western blot, high-content imaging

  • Localization studies: IF with FITC-conjugated TSC2 antibody

  • Interaction analysis: Co-IP, PLA, FRET

  • Functional assays: Cap-dependent translation, autophagy flux

What controls should be included when using phospho-specific TSC2 antibodies?

Robust experimental design with phospho-specific TSC2 antibodies requires comprehensive controls:

• Positive controls:

  • Cells treated with known pathway activators (e.g., insulin for Thr1462 phosphorylation)

  • Overexpression of constitutively active upstream kinases

  • Positive cell lines with high basal phosphorylation levels

• Negative controls:

  • Phosphatase treatment of lysates/samples

  • Pathway inhibitor treatment (kinase inhibitors)

  • Phospho-null mutants (specific residue replaced with alanine)

  • siRNA/shRNA knockdown of TSC2

• Antibody specificity controls:

  • Peptide competition assays with phosphorylated vs. non-phosphorylated peptides

  • Comparison of multiple phospho-specific antibodies targeting different epitopes

  • Parallel detection with phospho-independent TSC2 antibody

• Technical controls:

  • Loading controls (total protein stain, housekeeping proteins)

  • Transfer efficiency controls for Western blot

  • Isotype control antibodies for background estimation

  • Single-color controls for fluorescence microscopy

• Biological validation:

  • Genetic modification of the phosphorylation site

  • Correlation with functional readouts (mTORC1 activity)

  • Pharmacological manipulation of relevant kinases/phosphatases