TSC2 Antibody, Biotin conjugated

What is TSC2 and what is its significance in cellular signaling pathways?

TSC2, also known as Tuberin or Tuberous Sclerosis 2 protein, is a tumor suppressor that forms a functional complex with TSC1 (Hamartin). This TSC1-TSC2 complex plays a critical role in regulating the mammalian target of rapamycin complex 1 (TORC1), which coordinates nutritional, hormonal, and other cellular signals to regulate the cellular growth machinery. Inactivation of the TSC1-TSC2 complex results in inappropriate TORC1 activity and cellular growth defects . The N-terminal region of TSC2 is particularly important for interaction with TSC1, while the C-terminal region contains other functional domains essential for TSC2 activity .

What are the primary applications for TSC2 Antibody, Biotin conjugated in experimental protocols?

TSC2 Antibody, Biotin conjugated has several validated research applications:

• Western Blot analysis: For detecting and quantifying TSC2 protein expression in cell and tissue lysates

• Immunocytochemistry/Immunofluorescence: For visualizing TSC2 subcellular localization

• ELISA: For quantitative detection of TSC2 in solution-based assays

The biotin conjugation enables signal amplification through streptavidin-based detection systems, which is particularly valuable when studying proteins with lower expression levels or when enhanced sensitivity is required .

What species cross-reactivity can researchers expect from commercially available TSC2 Antibody, Biotin conjugated?

According to product specifications, commercially available TSC2 Antibody, Biotin conjugated (such as NBP1-76619B) demonstrates confirmed reactivity with human and mouse TSC2 . Rat reactivity may be predicted based on sequence homology analysis, with the immunogen displaying approximately 86% sequence identity with rat TSC2 . When working with species not explicitly validated, researchers should perform preliminary validation experiments and consider that cross-reactivity is largely dependent on epitope conservation across species.

What is the optimal sample preparation protocol for detecting TSC2 using Biotin-conjugated antibodies?

For optimal TSC2 detection, consider the following sample preparation protocol:

• Cell lysis: Use a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktail

• Phosphorylation studies: Include phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) when studying TSC2 phosphorylation status

• Protein quantification: Determine protein concentration using Bradford or BCA assay

• Sample denaturation: Heat samples to 95°C for 5 minutes in Laemmli buffer for Western blotting

• Loading control: Include analysis of housekeeping proteins to normalize for loading variations

For immunofluorescence applications, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 is generally effective for TSC2 detection .

How should researchers optimize blocking conditions when using TSC2 Antibody, Biotin conjugated?

Due to the biotin conjugation, special consideration must be given to blocking conditions:

• Avoid avidin-biotin blocking kits when using direct detection methods

• Use 5% BSA in TBS-T rather than milk-based blockers to reduce background

• Consider adding 0.1% Tween-20 to all washing and antibody dilution buffers

• When working with tissues containing endogenous biotin (liver, kidney, brain), implement specific endogenous biotin blocking steps before antibody application

• Titrate blocking reagent concentration (3-5%) to optimize signal-to-noise ratio

Optimal dilution of the TSC2 Antibody, Biotin conjugated should be experimentally determined for each application and may range from 1:500 to 1:2000 depending on the specific protocol and detection system .

What detection systems are most compatible with TSC2 Antibody, Biotin conjugated?

For Western blot applications, streptavidin-HRP systems typically provide excellent sensitivity, while fluorophore-conjugated streptavidin delivers superior results for immunofluorescence microscopy .

How can TSC2 Antibody, Biotin conjugated be utilized to study the TSC1-TSC2 complex formation?

For investigating TSC1-TSC2 complex formation, researchers can employ the following approaches using TSC2 Antibody, Biotin conjugated:

• Co-immunoprecipitation: Use the antibody to pull down TSC2 and detect co-precipitated TSC1

• Proximity ligation assay (PLA): Combine the biotinylated TSC2 antibody with an antibody against TSC1 to visualize protein-protein interactions in situ

• FRET analysis: Utilize the biotin-conjugated TSC2 antibody with fluorophore-labeled streptavidin paired with a differently labeled TSC1 antibody

• Immunofluorescence co-localization: Assess the spatial overlap of TSC1 and TSC2 signals

Research has demonstrated that the N-terminal region of TSC2 (amino acids 1-900) is crucial for interaction with TSC1, with TSC1 expression levels being reduced in the presence of pathogenic TSC2 variants with amino acid changes in this region . The biotinylated antibody can be particularly useful for studying these interaction dynamics when the immunogen is located within the first 50 amino acids of TSC2 .

What strategies can address non-specific binding issues when using TSC2 Antibody, Biotin conjugated?

When encountering non-specific binding with TSC2 Antibody, Biotin conjugated, implement these troubleshooting strategies:

• Increase washing stringency: Use higher salt concentration (up to 500 mM NaCl) in wash buffers

• Optimize antibody concentration: Perform titration experiments to determine the minimal effective concentration

• Pre-adsorb the antibody: Incubate with non-relevant tissue lysate before application to target samples

• Add competing proteins: Include 0.1-0.5% BSA or 0.1-0.5% non-fat dry milk in antibody diluent

• Use alternative blockers: Test casein, fish gelatin, or commercial blocking reagents

• Include detergent: Add 0.05-0.1% Tween-20 to reduce hydrophobic interactions

• Validate specificity: Run parallel experiments with peptide competition to confirm specific binding

These approaches should be systematically tested to determine the optimal conditions for your specific experimental system .

How does the epitope location of TSC2 Antibody, Biotin conjugated affect its utility in detecting disease-related TSC2 variants?

The epitope location significantly impacts the antibody's ability to detect disease-associated variants:

• N-terminal epitope antibodies (such as NBP1-76619B, targeting within the first 50 amino acids) may detect most missense mutations but might not recognize truncated proteins resulting from nonsense mutations or frameshift mutations beyond the epitope region .

• For comprehensive detection of pathogenic variants:

  • Multiple antibodies targeting different domains should be employed

  • Both N- and C-terminal targeted antibodies provide complementary information

  • Domain-specific antibodies help characterize functional consequences of mutations

• Epitope accessibility considerations:

  • Some mutations may alter protein folding, potentially masking or exposing certain epitopes

  • Post-translational modifications near the epitope may affect antibody binding

  • Protein-protein interactions could sterically hinder epitope recognition

Research on 107 TSC2 variants (69 pathogenic and 38 probably neutral) revealed that many disease-causing mutations affect protein function without eliminating expression, highlighting the importance of epitope selection when studying variant proteins .

How can TSC2 Antibody, Biotin conjugated contribute to understanding mTOR pathway regulation?

TSC2 Antibody, Biotin conjugated can be instrumental in elucidating mTOR pathway regulation through:

• Phosphorylation-specific analysis: Detecting TSC2 phosphorylation status at key regulatory sites

• Subcellular localization studies: Tracking TSC2 translocation in response to pathway activation/inhibition

• Protein complex formation: Examining TSC2 interactions with TSC1 and other pathway components

• Functional readouts: Correlating TSC2 levels/activity with downstream markers like S6K phosphorylation

• Inhibitor studies: Assessing pathway response to rapamycin and other mTOR inhibitors

Studies have shown that inactivation of the TSC1-TSC2 complex results in inappropriate TORC1 activity and cell growth defects. The complex functions as a critical negative regulator of mTOR signaling, integrating multiple upstream signals. Researchers have used TSC2 antibodies to quantify the effects of different TSC2 variants on TSC1 expression levels and on T389 phosphorylation of S6K, a downstream target of TORC1 .

What controls should be included when using TSC2 Antibody, Biotin conjugated for quantitative protein analysis?

For Western blot quantification, a standard curve using recombinant TSC2 protein at known concentrations can enable absolute quantification. For relative quantification, normalization to housekeeping proteins is essential to control for loading variations .

How should researchers interpret discrepancies between results obtained with different TSC2 antibodies?

When facing discrepancies between different TSC2 antibodies:

• Epitope differences: Map the epitope regions of each antibody and consider how various TSC2 domains might be differentially affected by experimental conditions or mutations

• Post-translational modifications: Consider whether modifications near the epitope may affect antibody binding

• Protein conformation: Different antibodies may recognize distinct conformational states of TSC2

• Isoform specificity: Verify whether antibodies recognize all or specific TSC2 isoforms

• Antibody validation: Review validation data for each antibody, including specificity testing and knockout controls

Studies analyzing TSC2 variants have shown that different regions of the protein are involved in distinct functions - the N-terminal region (amino acids 1-900) is primarily responsible for TSC1 interaction, while the C-terminal region (amino acids 900-1807) contains the functional GAP domain. Antibodies targeting different regions may therefore provide complementary information about TSC2 functionality .

What approaches can enhance detection sensitivity when using TSC2 Antibody, Biotin conjugated for low-abundance samples?

For detecting low-abundance TSC2 protein:

• Signal amplification strategies:

  • Employ tyramide signal amplification (TSA) to enhance fluorescence or chromogenic signals

  • Utilize poly-HRP or poly-biotin secondary detection systems

  • Consider using quantum dots conjugated to streptavidin for enhanced photostability

• Sample enrichment methods:

  • Perform immunoprecipitation prior to Western blotting

  • Use subcellular fractionation to concentrate TSC2 in relevant fractions

  • Consider using sensitive detection substrates (e.g., chemiluminescent with enhanced sensitivity)

• Protocol optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize blocking and washing conditions to minimize background

  • Use PVDF membranes with smaller pore size (0.2 μm) for Western blotting

• Digital enhancement:

  • Employ advanced imaging systems with high-sensitivity cameras

  • Use extended exposure times with integration of multiple images

  • Apply appropriate background subtraction algorithms

Researchers have improved immunoblot assays for TSC2 detection to enable more accurate analysis of larger numbers of variants with increased reproducibility and reduced cost .

How can TSC2 Antibody, Biotin conjugated be utilized in tuberous sclerosis complex (TSC) research?

TSC2 Antibody, Biotin conjugated offers several valuable applications in TSC research:

• Variant classification: Assess the functional consequences of unclassified TSC2 variants identified in patients

• Genotype-phenotype correlation: Compare biochemical effects of different pathogenic variants with corresponding patient phenotypes

• Therapeutic response monitoring: Evaluate changes in TSC2 expression or localization in response to mTOR inhibitors

• Biomarker studies: Investigate TSC2 as a potential prognostic or diagnostic marker

• Drug screening: Identify compounds that restore function of specific TSC2 mutants

What methodological considerations are important when studying TSC2 phosphorylation states?

When investigating TSC2 phosphorylation:

• Sample preparation:

  • Rapidly harvest cells to preserve phosphorylation status

  • Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in all buffers

  • Maintain samples at 4°C throughout processing

• Analytical approaches:

  • Use Phos-tag™ SDS-PAGE for enhanced separation of phosphorylated species

  • Consider phospho-specific antibodies for key regulatory sites

  • Combine with lambda phosphatase treatments as controls

  • Employ mass spectrometry for comprehensive phosphorylation mapping

• Stimulation conditions:

  • Standardize serum starvation protocols before stimulation

  • Use positive controls for pathway activation (insulin, EGF)

  • Include time-course experiments to capture transient phosphorylation events

• Data interpretation:

  • Quantify phosphorylation relative to total TSC2 levels

  • Consider multiple phosphorylation sites and their combinatorial effects

  • Correlate phosphorylation changes with functional readouts (e.g., mTORC1 activity)

Research has shown that TSC2 functions as part of a protein complex that integrates multiple growth factor- and energy-dependent signals to control cell growth. Phosphorylation plays a critical role in regulating TSC2 activity in response to these signals .