TSC2 Antibody, HRP conjugated

What is TSC2 and why is it a significant research target?

TSC2, also known as tuberin, functions primarily as a tumor suppressor protein. It plays critical roles in vesicular transport, cell growth regulation, and transcription mediated by steroid receptors. TSC2 forms a complex with TSC1 (hamartin) that facilitates vesicular docking and specifically stimulates the intrinsic GTPase activity of RAP1A and RAB5, suggesting a mechanism for regulating cellular growth . In humans, the canonical TSC2 protein consists of 1807 amino acid residues with a molecular mass of approximately 200.6 kDa . The protein's significance in research stems from its role as a negative regulator of mTORC1 signaling, inhibiting nutrient-mediated or growth factor-stimulated phosphorylation of S6K1 and EIF4EBP1 . Mutations in tuberin lead to constitutive activation of RAP1A in tumors, making it a crucial target for cancer research .

What are the advantages of HRP-conjugated TSC2 antibodies?

HRP-conjugated TSC2 antibodies offer significant advantages in immunodetection protocols:

When selecting HRP-conjugated TSC2 antibodies, researchers should consider the specific epitope recognized, clonality (polyclonal vs. monoclonal), and validation data for their intended application .

What applications are optimal for TSC2-HRP antibodies?

TSC2-HRP antibodies demonstrate versatility across multiple immunodetection techniques. Based on manufacturer recommendations, these antibodies perform optimally in the following applications with specified dilution ranges:

• Western Blot: 1:100-1000 dilution, ideal for detecting TSC2 protein expression levels and monitoring post-translational modifications

• Immunohistochemistry (paraffin): 1:100-500 dilution, suitable for localizing TSC2 in tissue sections

• Immunoprecipitation: Optimal dilutions should be experimentally determined

• ELISA: Typically effective at similar dilutions to Western blot applications

While some sources indicate that certain anti-TSC2 antibodies may not perform well in Western blot applications, this appears to be antibody-specific rather than a limitation of all TSC2-HRP conjugated antibodies . Researchers should conduct preliminary validation experiments to determine optimal conditions for their specific experimental system.

How should I optimize Western blot protocols for TSC2-HRP antibodies?

Optimizing Western blot protocols for TSC2-HRP antibodies requires careful consideration of several experimental parameters:

• Sample preparation:

  • Use RIPA buffer with protease and phosphatase inhibitors for comprehensive protein extraction

  • Include 1-2 mM EDTA to protect TSC2 from metalloproteases

  • Process samples rapidly at 4°C to prevent degradation

• Gel selection and transfer conditions:

  • Use 6-8% polyacrylamide gels to effectively resolve the 200.6 kDa TSC2 protein

  • Transfer to PVDF membranes (rather than nitrocellulose) for improved retention of high molecular weight proteins

  • Extend transfer time to 2 hours at 30V or use overnight transfer at 15V for complete transfer

• Antibody incubation:

  • Start with a 1:500 dilution for initial optimization

  • Incubate membranes at 4°C overnight for improved signal-to-noise ratio

  • Use 5% BSA in TBST rather than milk for blocking and antibody dilution to reduce background

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrate optimized for HRP detection

  • Begin with shorter exposure times (30 seconds) and increase as needed

  • Consider using a digital imaging system for more precise quantification

It's advisable to include both positive controls (cells/tissues known to express TSC2) and negative controls (TSC2-knockout or siRNA treated samples) to validate antibody specificity .

What controls are essential when working with TSC2-HRP antibodies?

Implementing appropriate controls is critical for ensuring reliable and interpretable results with TSC2-HRP antibodies:

For experiments examining TSC2 phosphorylation states, additional controls with mTOR pathway activators (insulin, EGF) or inhibitors (rapamycin, torin) can provide valuable functional validation . When studying TSC2 interactions with TSC1, co-immunoprecipitation experiments should include controls for detecting both proteins to confirm complex formation .

How can I validate TSC2-HRP antibody specificity for my research?

Comprehensive validation of TSC2-HRP antibody specificity involves multiple complementary approaches:

• Molecular weight verification:

  • Confirm detection of bands at the expected molecular weight (200.6 kDa for full-length TSC2)

  • Be aware that up to 8 different isoforms have been reported for TSC2

• Genetic manipulation:

  • Compare signal between wild-type and TSC2 knockdown/knockout samples

  • Observe signal reduction/elimination in genetically modified samples

• Epitope mapping:

  • Use antibodies targeting different TSC2 regions to confirm consistent detection

  • For example, compare results from antibodies recognizing N-terminal (residues 1-50) versus other domains

• Cross-species reactivity testing:

  • Verify reactivity with human, mouse, and rat TSC2 as claimed by manufacturers

  • Note any species-specific differences in detection sensitivity

• Phosphorylation-dependent recognition:

  • For phospho-specific antibodies, treat samples with phosphatases to confirm specificity

  • Use pathway modulators (e.g., mTOR inhibitors) to alter phosphorylation states

• Immunodepletion:

  • Sequentially immunoprecipitate TSC2 and analyze the depleted lysate

  • Complete disappearance of signal indicates high antibody specificity

Document all validation steps methodically and include representative images in supplementary materials for publications to enhance reproducibility of research findings .

How can TSC2-HRP antibodies be used to investigate mTOR signaling pathways?

TSC2-HRP antibodies offer powerful tools for investigating mTOR signaling pathways through multiple experimental approaches:

• Phosphorylation status analysis:

  • Monitor TSC2 phosphorylation at key regulatory sites (Ser939, Ser1254, Ser1387, Thr1462, Tyr1571)

  • Phosphorylation at these sites by AKT, AMPK, or ERK modulates TSC2 function in mTOR regulation

• TSC1-TSC2 complex formation:

  • Use co-immunoprecipitation with TSC2-HRP antibodies to isolate and detect the TSC1-TSC2 complex

  • Examine how pathway modulators affect complex stability and formation

• Subcellular localization studies:

  • Track TSC2 translocation between cytoplasm and membrane compartments in response to stimuli

  • Combine with markers for specific cellular compartments to determine precise localization

• Downstream effector activity:

  • Correlate TSC2 expression/phosphorylation with S6K1 and 4EBP1 phosphorylation status

  • Establish functional relationships between TSC2 activity and mTORC1 signaling output

• RhebGAP activity assessment:

  • Examine TSC2's GTPase-activating protein function toward Rheb

  • Monitor Rheb-GTP levels in relation to TSC2 activity modifications

When designing these experiments, researchers should consider using synchronized cell populations and carefully timed treatments to capture the dynamic nature of mTOR signaling events. The HRP conjugation enables sensitive detection of TSC2 in complex experimental systems without secondary antibody complications .

What approaches can distinguish between different TSC2 isoforms?

Distinguishing between the multiple TSC2 isoforms (up to 8 reported variants) requires strategic experimental approaches:

• Isoform-specific antibody selection:

  • Choose antibodies targeting regions that differ between isoforms

  • Verify epitope location relative to known splicing junctions

• Resolution optimization:

  • Use lower percentage acrylamide gels (5-6%) to better separate high molecular weight isoforms

  • Extend electrophoresis time to enhance band separation

• 2D gel electrophoresis:

  • Combine isoelectric focusing with SDS-PAGE to separate isoforms by both charge and size

  • HRP-conjugated antibodies work effectively in 2D Western blot detection

• Molecular techniques:

  • Complement protein detection with RT-PCR using isoform-specific primers

  • Compare results with antibody detection patterns to confirm isoform identity

• Mass spectrometry validation:

  • Use immunoprecipitation with TSC2 antibodies followed by mass spectrometry

  • Identify isoform-specific peptide sequences to confirm antibody selectivity

When reporting isoform detection, researchers should clearly document the specific antibody epitope and include positive controls expressing verified isoforms when possible. Different tissues may express distinct isoform profiles, with notable expression in caudate, thyroid gland, and adrenal gland tissues .

What are common issues with TSC2-HRP antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with TSC2-HRP antibodies:

For HRP-conjugated antibodies specifically, additional considerations include:

• Avoiding sodium azide in buffers as it inhibits HRP activity

• Protecting the antibody from light exposure

• Storing according to manufacturer recommendations (typically at 4°C in the dark)

When troubleshooting complex scenarios, consider running side-by-side comparisons with unconjugated TSC2 antibodies to determine if issues are related to the primary antibody specificity or the HRP conjugation .

How should inconsistent results between different TSC2 detection methods be interpreted?

When faced with discrepancies between different TSC2 detection methods, systematic analysis is essential:

• Antibody epitope considerations:

  • Different antibodies may target distinct TSC2 domains affected differently by protein conformation

  • Phosphorylation near epitopes can mask antibody binding sites

  • Some antibodies may preferentially recognize certain TSC2 isoforms

• Methodological differences:

  • Native vs. denatured conditions affect epitope accessibility

  • Fixation methods in IHC may alter protein structure and epitope availability

  • Western blot detects total protein while IHC provides spatial information

• Technical validation approach:

  • Use multiple antibodies recognizing different TSC2 epitopes

  • Compare different detection methods (fluorescence vs. colorimetric)

  • Validate with orthogonal techniques (mass spectrometry, RNA-seq)

• Biological interpretation:

  • Consider post-translational modifications affecting specific epitopes

  • Evaluate subcellular localization affecting detectability

  • Assess protein-protein interactions that might mask binding sites

When integrating conflicting data, weigh results based on the robustness of controls and validation steps performed. For publications, transparently report discrepancies and provide possible explanations rather than selecting only concordant results .

How are TSC2-HRP antibodies advancing cancer research?

TSC2-HRP antibodies are enabling significant advances in cancer research through several mechanisms:

• Diagnostic biomarker development:

  • Detection of altered TSC2 expression in tumor samples versus normal tissues

  • Correlation of TSC2 levels with tumor progression and patient outcomes

  • Potential for developing TSC2-based companion diagnostics for mTOR inhibitor therapies

• mTOR pathway dysregulation analysis:

  • Monitoring TSC2 loss/mutation effects on mTOR hyperactivation in various cancers

  • Evaluating TSC2 phosphorylation states in response to upstream oncogenic signals

  • Understanding resistance mechanisms to mTOR-targeted therapies

• Drug discovery applications:

  • Screening compounds that restore TSC2 function in mutant backgrounds

  • Monitoring TSC2 activity as a readout for drug efficacy

  • Identifying synthetic lethal interactions in TSC2-deficient contexts

• Tumor metabolism studies:

  • Investigating TSC2's role in metabolic reprogramming of cancer cells

  • Linking TSC2 function to Warburg effect and glutaminolysis pathways

  • Exploring metabolic vulnerabilities in TSC2-deficient tumors

Recent research has employed TSC2-HRP antibodies to examine constitutive activation of RAP1A in tumors resulting from tuberin mutations . Additionally, studies are investigating TSC2's interaction with the RAB5 GTPase pathway, suggesting new therapeutic approaches targeting vesicular trafficking in cancer cells .

What emerging applications exist for TSC2-HRP antibodies in neurodevelopmental disorders?

TSC2-HRP antibodies are increasingly being applied to study neurodevelopmental disorders, particularly tuberous sclerosis complex (TSC):

• Neuropathological analyses:

  • Characterizing TSC2 expression patterns in cortical tubers and subependymal nodules

  • Correlating TSC2 deficiency with abnormal neuronal morphology and migration

  • Examining mosaicism in TSC2 expression within brain tissues

• Neuronal signaling studies:

  • Investigating TSC2's role in neuronal polarity and axon guidance

  • Monitoring local protein synthesis regulation in dendritic spines

  • Exploring synapse formation and plasticity mechanisms

• Preclinical therapy evaluation:

  • Assessing restoration of TSC2 function after genetic or pharmacological interventions

  • Monitoring downstream mTOR pathway normalization in neuronal models

  • Evaluating efficacy of combination therapies targeting multiple nodes of the pathway

• Neuronal-glial interaction research:

  • Examining cell-type specific TSC2 functions in neurons versus glia

  • Investigating non-cell autonomous effects of TSC2 deficiency

  • Studying neuroinflammatory responses in TSC2-deficient brain regions

These approaches are facilitated by the direct detection capabilities of HRP-conjugated antibodies, which allow for sensitive visualization of TSC2 in complex neural tissues without additional amplification steps. The ability to detect TSC2 in both human and model organism tissues (mouse, rat) enables translational research between preclinical models and clinical samples .

TSC2-HRP antibodies are proving valuable in understanding how mTOR dysregulation contributes to epileptogenesis, autism spectrum features, and cognitive impairment in TSC and related neurodevelopmental disorders.