CASTOR2 Antibody, Biotin conjugated

What is CASTOR2 and how does it differ from CASTOR1?

CASTOR2 (previously known as GATSL2 or GATS-like protein 2) is a 329-amino acid protein with a molecular weight of approximately 36.1 kDa that functions as a cytosolic component in the mTORC1 signaling pathway . While CASTOR1 and CASTOR2 share significant sequence homology (63% identity), they exhibit crucial functional differences:

• Structure: Both contain ACT domains, but CASTOR2's ACT domains interact constitutively with each other, whereas CASTOR1's ACT domains interact in an arginine-dependent manner .

• Binding properties: CASTOR2 binds more strongly to GATOR2 than CASTOR1 homodimers, and this binding is largely insensitive to amino acid availability .

• Arginine sensing: Unlike CASTOR1, CASTOR2 lacks arginine-binding capability. This difference appears to be partly due to variations in residues along the ACT2-ACT4 interface (particularly at positions 108-110) .

• Complex formation: Both proteins can form homodimers and heterodimers (CASTOR1-CASTOR2), with differing binding affinities to GATOR2 .

These differences suggest complementary roles in the regulation of mTORC1 signaling, with CASTOR1 functioning as the arginine sensor while CASTOR2 potentially serving other regulatory functions.

How does CASTOR2 function in the mTORC1 signaling pathway?

CASTOR2 participates in the mTORC1 signaling pathway as a negative regulator through its interaction with the GATOR complex . The current understanding suggests:

• CASTOR2 homodimers bind more strongly to GATOR2 than CASTOR1 homodimers .

• While CASTOR1-GATOR2 interaction is strongly regulated by arginine availability, CASTOR2-GATOR2 binding remains relatively constant regardless of amino acid conditions .

• When CASTOR1 is depleted, CASTOR2-GATOR2 interaction becomes completely insensitive to amino acid availability .

• CASTOR2 forms heterodimers with CASTOR1, which show intermediate binding strength to GATOR2 and retain arginine sensitivity .

This suggests that CASTOR2 may function as a constitutive inhibitor of GATOR2, potentially setting a baseline for pathway inhibition, while CASTOR1 provides arginine-responsive modulation of this inhibition.

What is the scientific principle behind biotin conjugation and why is it useful for CASTOR2 antibodies?

Biotin conjugation leverages the exceptionally high affinity between biotin (Vitamin H) and avidin/streptavidin proteins for enhanced detection in immunoassays:

• Chemical basis: Biotin is covalently attached to antibodies through various chemical conjugation strategies that target amino groups on the antibody .

• Binding mechanism: The biotin-avidin/streptavidin interaction has one of the strongest non-covalent bonds in nature (Ka ≈ 10^15 M^-1), making it extremely stable and specific .

• Signal amplification: Multiple biotin molecules can be conjugated to a single antibody, allowing for binding of multiple streptavidin molecules, each potentially carrying detection enzymes or fluorophores .

For CASTOR2 antibodies specifically, biotin conjugation offers:

• Enhanced sensitivity for detecting low-abundance CASTOR2 protein in complex samples

• Flexibility in detection methods (can be paired with various streptavidin conjugates)

• Compatibility with multiple immunoassay techniques including Western blot, ELISA, and immunohistochemistry

How does Biotin-SP differ from standard biotin conjugation, and when should it be used?

Biotin-SP refers to biotin with a 6-atom spacer positioned between the biotin molecule and the antibody to which it is conjugated:

Biotin-SP should be used when:

• Maximum sensitivity is required for detecting low-abundance targets like CASTOR2

• Working with streptavidin conjugates that may benefit from improved spatial access to biotin

• Performing enzyme immunoassays, especially those using alkaline phosphatase detection systems

The spacer "extends the biotin moiety away from the antibody surface, making it more accessible to binding sites on streptavidin" , which enhances detection efficiency.

What are the optimal conditions for using CASTOR2 antibody, biotin conjugated in Western blot applications?

For optimal Western blot results with biotin-conjugated CASTOR2 antibodies:

Protocol optimization:

• Sample preparation: Ensure complete cell lysis using buffers containing phosphatase inhibitors to preserve CASTOR2 phosphorylation status

• Protein loading: Load 20-30 μg total protein per lane (may require optimization for your specific sample)

• Transfer conditions: Use PVDF membrane for higher protein binding capacity

• Blocking: 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution: 1:1000-1:2000 (optimize for your specific antibody)

  • Incubation: Overnight at 4°C or 2 hours at room temperature

• Detection system: Use streptavidin-HRP (1:5000-1:10000) or streptavidin-AP (1:2000-1:5000) for 1 hour at room temperature

Expected results:

• CASTOR2 should appear as a band at approximately 36.1 kDa

• Positive controls: Use lysates from human cells known to express CASTOR2

• Negative controls: Consider using CASTOR2 knockout cells or CASTOR2-depleted samples through RNAi

Troubleshooting recommendations:

• If signal is weak, consider longer incubation with the detection reagent or higher antibody concentration

• If background is high, increase washing steps and optimize blocking conditions

• For cross-reactivity concerns, use pre-adsorbed secondary reagents

How should researchers optimize ELISA protocols when using biotin-conjugated CASTOR2 antibodies?

For ELISA applications with biotin-conjugated CASTOR2 antibodies:

Protocol considerations:

• Coating: If using a sandwich ELISA, coat plates with a capture antibody against CASTOR2 (non-biotinylated) at 1-5 μg/mL in carbonate buffer (pH 9.6)

• Blocking: 1-3% BSA in PBS (ensure the BSA is biotin-free to avoid interference)

• Sample preparation: Prepare cell lysates or serum samples in appropriate dilution buffers

• Biotin-conjugated antibody application:

  • Starting dilution: 1:2000-1:4000

  • Incubation: 1-2 hours at room temperature with gentle shaking

• Detection: Use streptavidin-HRP conjugate (1:5000-1:10000 dilution)

• Development: TMB substrate for HRP with appropriate stop solution

Optimization parameters:

• Antibody titration: Test serial dilutions of the biotin-conjugated CASTOR2 antibody to determine optimal concentration

• Incubation times: Vary from 30 minutes to overnight at 4°C to find the best signal-to-noise ratio

• Temperature effects: Compare room temperature vs. 37°C vs. 4°C incubations

• Buffer composition: Test different detergent concentrations and pH values

Standard curve preparation:
If quantifying CASTOR2, use recombinant CASTOR2 protein at concentrations ranging from 0-1000 ng/mL to establish a standard curve.

How can researchers distinguish between true CASTOR2 signal and non-specific binding when using biotin-conjugated antibodies?

Non-specific binding can confound results when using biotin-conjugated antibodies. Here are methodological approaches to distinguish true CASTOR2 signal:

Controls to implement:

• Negative control samples:

  • CASTOR2 knockout/knockdown cells or tissues

  • Use of competitive blocking peptide specific to the CASTOR2 epitope

  • Omission of primary antibody while maintaining all other detection reagents

• Specificity controls:

  • Pre-adsorption controls: Incubate antibody with recombinant CASTOR2 protein prior to application

  • Cross-reactivity assessment with CASTOR1 (63% sequence identity)

  • Validation with alternative CASTOR2 antibodies targeting different epitopes

• Endogenous biotin blocking:

  • Pre-block samples with avidin/streptavidin followed by biotin to neutralize endogenous biotin

  • Use commercial biotin blocking kits before applying biotin-conjugated antibodies

Analytical approaches:

• Molecular weight verification: CASTOR2 should appear at approximately 36.1 kDa

• Observe expected subcellular localization (cytoplasmic)

• Compare staining patterns with published literature and database information

Validation methods:

• Parallel analysis with non-biotinylated CASTOR2 antibodies using direct detection methods

• Confirmation by orthogonal techniques (mass spectrometry, RNA expression analysis)

• Biological validation through functional assays of the mTORC1 pathway

What are the most common technical issues when working with CASTOR2 antibody, biotin conjugated, and how can they be resolved?

Researchers commonly encounter several technical challenges when working with biotin-conjugated CASTOR2 antibodies:

Issue 1: High background signal

• Causes: Endogenous biotin in samples; insufficient blocking; excessive antibody concentration

• Solutions:

  • Implement avidin/biotin blocking steps before applying primary antibody

  • Optimize blocking conditions (test different blocking agents: BSA, casein, commercial blockers)

  • Titrate antibody to lower concentrations

  • Increase wash duration and frequency between steps

Issue 2: Weak or absent signal

• Causes: Protein degradation; inefficient conjugation; epitope masking; low CASTOR2 expression

• Solutions:

  • Include protease inhibitors in sample preparation

  • Verify antibody quality with dot blot testing

  • Try different epitope retrieval methods for fixed samples

  • Concentrate samples for low-abundance targets

  • Increase antibody concentration or incubation time

Issue 3: Multiple bands or unexpected band sizes in Western blots

• Causes: Protein degradation; post-translational modifications; antibody cross-reactivity

• Solutions:

  • Verify using recombinant CASTOR2 as positive control

  • Test in CASTOR2 knockout/knockdown samples

  • Use fresher samples with added protease inhibitors

  • Compare with known CASTOR2 interacting proteins (CASTOR1, GATOR2 components)

Issue 4: Inconsistent results between experiments

• Causes: Antibody degradation; variable sample quality; protocol inconsistencies

• Solutions:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Standardize lysate preparation

  • Implement detailed protocol documentation

  • Include consistent positive controls across experiments

How can researchers use biotin-conjugated CASTOR2 antibodies to study CASTOR1/CASTOR2 heterodimer formation and its impact on mTORC1 signaling?

Studying CASTOR1/CASTOR2 heterodimer formation requires sophisticated approaches leveraging biotin-conjugated antibodies:

Experimental design strategies:

• Co-immunoprecipitation (Co-IP) with differential tagging:

  • Use biotin-conjugated anti-CASTOR2 antibodies for pulldown experiments

  • Detect associated CASTOR1 using non-biotinylated anti-CASTOR1 antibodies

  • Analyze co-precipitated proteins under various amino acid conditions (particularly arginine)

• Proximity ligation assay (PLA):

  • Use biotin-conjugated anti-CASTOR2 plus non-biotinylated anti-CASTOR1

  • Apply streptavidin-linked PLA probe and appropriate secondary antibody-linked PLA probe

  • Visualize interaction signals at subcellular resolution

• Sequential immunoprecipitation:

  • First IP: Anti-CASTOR1 antibody to capture all CASTOR1-containing complexes

  • Second IP: Biotin-conjugated anti-CASTOR2 antibody to isolate heterodimers specifically

  • Western blot to confirm identity and quantify relative amounts

Analysis of physiological relevance:

• Compare heterodimer formation under arginine-rich vs. arginine-starved conditions

• Assess interaction with GATOR2 components (mios, WDR24, WDR59)

• Measure downstream mTORC1 activity (phospho-S6K, phospho-4E-BP1)

Mechanistic insights:
Research has shown that "CASTOR1-CASTOR2 heterodimer bound to GATOR2 at an intermediate level" between CASTOR1 and CASTOR2 homodimers, suggesting a regulatory role for heterodimer formation . The heterodimer retains arginine sensitivity, indicating that CASTOR1 properties dominate in this complex for arginine sensing .

What are the latest methodological advances for using biotin-conjugated antibodies to characterize the structural differences between CASTOR1 and CASTOR2 ACT domains?

Recent methodological advances have enhanced our ability to investigate the structural differences between CASTOR1 and CASTOR2 ACT domains:

Advanced structural biology approaches:

• Crosslinking mass spectrometry (XL-MS) with biotin-tagged antibodies:

  • Use biotin-conjugated domain-specific antibodies for targeted isolation

  • Apply chemical crosslinkers to stabilize protein interactions

  • Perform streptavidin-based purification followed by mass spectrometry

  • Identify interaction interfaces between ACT domains

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Use biotin-conjugated antibodies to purify CASTOR proteins

  • Monitor conformational changes upon arginine binding to CASTOR1 vs. CASTOR2

  • Map solvent accessibility differences between the paralogs

• Single-molecule FRET analysis with biotin-based immobilization:

  • Immobilize proteins via biotin-streptavidin interaction

  • Monitor conformational changes in real-time

  • Compare CASTOR1 arginine-induced conformational changes to CASTOR2

Key structural differences identified:
Research has identified critical regions that distinguish CASTOR1 and CASTOR2:

• Residues along the ACT2-ACT4 interface (positions 108-110) differ between CASTOR1 and CASTOR2

• CASTOR1 ACT domains interact in an arginine-dependent manner, whereas CASTOR2 ACT domains interact constitutively

• The β14-loop in CASTOR1 is involved in arginine binding and undergoes ordering upon arginine binding

These differences explain why "arginine binding orders the glycine-rich β14-loop to enable the intramolecular association of ACT2 and ACT4" in CASTOR1 but not in CASTOR2 .

How can researchers leverage biotin-conjugated CASTOR2 antibodies for high-resolution imaging of CASTOR2 localization relative to mTORC1 pathway components?

Biotin-conjugated CASTOR2 antibodies enable sophisticated imaging approaches to visualize CASTOR2 in relation to other mTORC1 components:

Advanced imaging methodologies:

• Super-resolution microscopy with biotin-streptavidin amplification:

  • Apply biotin-conjugated CASTOR2 antibodies to fixed cells

  • Use fluorophore-conjugated streptavidin (Alexa Fluor dyes)

  • Implement STORM, PALM, or STED imaging for resolution below diffraction limit

  • Achieve 20-50nm resolution of CASTOR2 localization

• Multi-color co-localization analysis:

  • Combine biotin-conjugated CASTOR2 antibodies with directly labeled antibodies against:

    • GATOR2 components (mios, WDR24, WDR59)

    • mTORC1 components (mTOR, raptor)

    • Lysosomal markers (LAMP1/2)

  • Analyze degree of co-localization under various nutrient conditions

• Live-cell imaging with split-biotin complementation:

  • Express CASTOR2 fused to a split-biotin tag

  • Express potential interaction partners with complementary split-biotin fragment

  • Apply cell-permeable streptavidin-fluorophore conjugates

  • Monitor dynamic interactions in response to nutrient availability

Analytical approaches:

• Pearson's correlation coefficient and Manders' overlap coefficient for co-localization quantification

• Single-particle tracking for dynamic interaction analysis

• 3D reconstruction to understand spatial organization within the cell

Expected findings:
Based on current understanding, researchers should observe:

• Primarily cytoplasmic localization of CASTOR2

• Dynamic association with GATOR2 components

• Potential redistribution patterns in response to cellular stress or nutrient availability

This high-resolution spatial information can provide new insights into how the physical organization of these components contributes to mTORC1 pathway regulation.

How might biotin-conjugated CASTOR2 antibodies be used to investigate potential therapeutic targeting of the mTORC1 pathway in disease models?

Biotin-conjugated CASTOR2 antibodies can facilitate several innovative approaches for therapeutic research:

Drug discovery applications:

• High-throughput screening platforms:

  • Develop ELISA-based assays using biotin-conjugated CASTOR2 antibodies to screen for compounds that modulate CASTOR2-GATOR2 interactions

  • Create bead-based multiplex assays to simultaneously monitor multiple mTORC1 pathway components

  • Establish cell-based reporter systems with biotin-based detection elements

• Target validation in disease models:

  • Use biotin-conjugated antibodies for immunohistochemical analysis of CASTOR2 expression in cancer tissues, neurodegenerative diseases, and metabolic disorders

  • Correlate CASTOR2 expression/localization with disease progression and treatment response

  • Perform comparative studies across different model systems

• Therapeutic monitoring:

  • Develop assays to monitor CASTOR2-related biomarkers during experimental therapies

  • Create companion diagnostic approaches using biotin-amplification systems

Disease-specific research applications:

• Cancer research: Investigate CASTOR2 expression in tumors with hyperactive mTORC1 signaling

• Neurodegenerative diseases: Examine CASTOR2 function in protein homeostasis and autophagy regulation

• Metabolic disorders: Study CASTOR2's role in nutrient sensing and metabolic adaptation

Research in these areas could lead to novel therapeutic strategies targeting specific nodes in the mTORC1 pathway, with CASTOR2 potentially serving as either a direct target or a biomarker for treatment efficacy.

What are the emerging techniques for studying post-translational modifications of CASTOR2 using biotin-conjugated antibodies?

Emerging techniques leveraging biotin-conjugated antibodies enable sophisticated analysis of CASTOR2 post-translational modifications:

Advanced methodologies:

• BioID or TurboID proximity labeling:

  • Express CASTOR2 fused to a biotin ligase (BioID2 or TurboID)

  • Identify proteins in close proximity that may regulate CASTOR2 through modifications

  • Use biotin-conjugated CASTOR2 antibodies for validation studies

• Phospho-specific detection systems:

  • Develop antibodies against predicted phosphorylation sites on CASTOR2

  • Use biotin-conjugated secondary antibodies with enhanced sensitivity

  • Employ multiplexed detection to monitor multiple modification sites simultaneously

• Mass spectrometry approaches:

  • Immunoprecipitate CASTOR2 using biotin-conjugated antibodies

  • Analyze with liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Map comprehensive modification landscape (phosphorylation, ubiquitination, acetylation)

  • Compare modifications under different cellular conditions

Integrated analysis frameworks:

• Correlate modifications with functional outcomes in the mTORC1 pathway

• Develop computational models predicting how modifications alter CASTOR2 structure and function

• Compare modification patterns between CASTOR1 and CASTOR2 to identify functional divergence

While specific CASTOR2 modifications are still being characterized, these approaches will help elucidate how post-translational regulation contributes to CASTOR2 function in the mTORC1 pathway. This knowledge could reveal new regulatory mechanisms and potential therapeutic targets.