TBC1D25 Antibody, HRP conjugated

What is TBC1D25 and what cellular functions does it regulate?

TBC1D25 (also known as OATL1) is a protein with a TBC domain that functions as a Rab GTPase-activating protein specific for RAB33B . It plays a crucial role in regulating autophagosome maturation, particularly in the fusion of autophagosomes with endosomes and lysosomes . Recent research has also identified TBC1D25 as an important regulator in cardiac remodeling through the TAK1 signaling pathway . Despite its previous name (ornithine aminotransferase-like 1), it has no actual similarity to ornithine aminotransferase . The protein contains specific regions that facilitate its interactions with other proteins, such as amino acids 138-226 in the C-terminal region, which are required for direct interaction with TAK1 .

What are the key specifications of commercially available TBC1D25 Antibody, HRP conjugated?

Commercial TBC1D25 Antibody, HRP conjugated preparations typically have the following specifications:

These antibodies are designed for research applications, with validation primarily for ELISA techniques .

Why is HRP conjugation used for TBC1D25 antibodies?

HRP (Horseradish Peroxidase) conjugation provides several advantages for antibody applications, particularly in immunoassays. The enzyme serves as a reporter molecule that catalyzes a colorimetric, chemiluminescent, or fluorescent reaction when appropriate substrates are added, allowing for visual or instrumental detection of the antibody binding . For TBC1D25 research, HRP conjugation enables direct detection in ELISA and other immunoassay formats without requiring secondary antibodies, simplifying experimental protocols and potentially reducing background signal. The chemical conjugation creates a stable, covalent linkage between the enzyme and antibody while preserving the functionality of both components .

What are the optimal assay conditions for TBC1D25 Antibody, HRP conjugated in ELISA?

When designing ELISA experiments with TBC1D25 Antibody, HRP conjugated, researchers should consider these methodological considerations:

• Antibody Dilution: Optimized protocols with lyophilized HRP-conjugated antibodies can work at dilutions as high as 1:5000, whereas traditional conjugation methods may only allow dilutions of 1:25 .

• Buffer Conditions: Use standard ELISA buffers (coating buffer: carbonate-bicarbonate buffer pH 9.6; blocking buffer: 1-3% BSA in PBS; washing buffer: PBS with 0.05% Tween-20).

• Incubation Parameters: For antigen coating, incubate at 4°C overnight or 37°C for 1-2 hours. For antibody incubation, 1-2 hours at room temperature is typically sufficient.

• Substrate Selection: TMB (3,3',5,5'-tetramethylbenzidine) is commonly used for HRP detection with absorbance reading at 450nm after stopping the reaction with 2N H₂SO₄.

• Controls: Include wells without primary antibody and wells without target antigen to assess specificity and background signal.

Researchers should perform antibody titration experiments to determine the optimal concentration for their specific experimental conditions, as sensitivity and background can vary based on target abundance and sample type.

How can TBC1D25 Antibody, HRP conjugated be used to study autophagy regulation?

TBC1D25 plays a critical role in autophagosome maturation through its interaction with RAB33B . To study this process:

• Autophagy Induction Models: Treat cells with rapamycin, starvation media, or other autophagy inducers.

• Co-localization Studies: Combine TBC1D25 antibody detection with markers for autophagosomes (LC3-II) and lysosomes (LAMP1) in immunofluorescence or flow cytometry.

• Protein Interaction Assays: Use the TBC1D25 antibody in co-immunoprecipitation experiments to examine interactions with RAB33B or other autophagy regulators.

• Quantification Approaches:

  • Western blotting to assess autophagy marker levels (LC3-II/LC3-I ratio)

  • Flow cytometry to measure autophagic flux

  • ELISA to quantify TBC1D25 levels under various conditions

• Functional Assays: Compare autophagy processes in models with TBC1D25 knockdown/knockout versus controls, using the antibody to confirm expression changes.

The HRP-conjugated format is particularly useful for quantitative ELISA assays to measure TBC1D25 expression levels under different experimental conditions affecting autophagy.

How does TBC1D25 participate in cardiac remodeling pathways?

TBC1D25 plays a significant role in cardiac remodeling through its interaction with the TAK1-JNK/p38 signaling pathway . Methodological approaches to study this function include:

• Cardiac Hypertrophy Models:

  • In vivo: Transverse aortic constriction (TAC) in TBC1D25-knockout and wild-type mice

  • In vitro: Angiotensin II treatment of cardiomyocytes with TBC1D25 overexpression or knockdown

• Cardiac Function Assessment:

  • Echocardiography for parameters like left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), ejection fraction (EF%), and fractional shortening (FS%)

  • Heart weight to body weight ratio (HW/BW) and heart weight to tibia length ratio (HW/TL) measurements

• Histological Analysis:

  • H&E staining for cardiomyocyte cross-sectional area

  • Picrosirius red staining for fibrosis assessment

• Molecular Pathway Analysis:

  • Western blotting for phosphorylation levels of TAK1, JNK, and p38

  • qRT-PCR for hypertrophy markers (ANP, BNP, MYH7) and fibrosis markers (Collagen Iα, Collagen III, CTGF)

Research has demonstrated that TBC1D25 knockout exacerbates cardiac hypertrophy, fibrosis, and dysfunction in TAC models, while TBC1D25 overexpression alleviates Angiotensin II-induced hypertrophy in vitro . The protective mechanism involves direct interaction between TBC1D25 and TAK1, requiring amino acids 138-226 in the C-terminal region of TBC1D25 and amino acids 1-300 in the C-terminal region of TAK1 .

What protein-protein interaction studies can be performed using TBC1D25 Antibody, HRP conjugated?

TBC1D25 Antibody, HRP conjugated can be utilized in several advanced protein-protein interaction study methodologies:

• Co-Immunoprecipitation (Co-IP) with Direct Detection:

  • Immunoprecipitate with a non-HRP conjugated antibody against the interaction partner

  • Detect TBC1D25 directly in the precipitate using the HRP-conjugated antibody

  • This approach eliminates the need for secondary antibodies in Western blotting

• Pull-Down Assays:

  • GST pull-down assays have confirmed direct interaction between TBC1D25 and TAK1

  • The HRP-conjugated antibody can provide direct detection of TBC1D25 in these assays

• ELISA-Based Interaction Studies:

  • Coat plates with purified interaction partner (e.g., TAK1)

  • Detect binding of TBC1D25 using the HRP-conjugated antibody

  • Quantify interaction strength under different conditions

• Protein Domain Mapping:

  • Use truncated constructs of TBC1D25 or interaction partners

  • Determine which domains are essential for interaction

  • Existing research has shown that amino acids 138-226 in TBC1D25 are critical for TAK1 interaction

• Competitive Binding Assays:

  • Use peptides or small molecules to disrupt specific interactions

  • Measure changes in binding using the HRP-conjugated antibody

These approaches can provide valuable insights into the mechanisms by which TBC1D25 regulates autophagy and cardiac remodeling through specific protein-protein interactions.

How can you optimize TBC1D25 Antibody, HRP conjugated performance through lyophilization?

Lyophilization can significantly enhance the performance of HRP-conjugated antibodies, including TBC1D25 antibodies. The methodological approach includes:

• Modified Conjugation Protocol:

  • Activate HRP using sodium meta-periodate to generate aldehyde groups by oxidizing carbohydrate moieties

  • Lyophilize the activated HRP before mixing with antibodies (1 mg/ml concentration)

  • Complete the conjugation process according to standard protocols

• Verification of Conjugation Success:

  • UV-Spectroscopy to confirm chemical modification

  • SDS-PAGE to verify the molecular weight shift indicative of successful conjugation

  • ELISA titration to determine functional activity

• Performance Assessment:

  • Compare lyophilized versus traditional conjugation methods at various dilutions

  • Evaluate signal-to-noise ratios and sensitivity limits

  • Research indicates lyophilized conjugates can be used at dilutions up to 1:5000, while traditional methods may only work at 1:25 dilutions

• Storage Optimization:

  • Determine if the lyophilized conjugate has enhanced stability

  • Test performance after various storage durations and conditions

This optimization strategy can lead to significant cost savings and improved experimental sensitivity when working with TBC1D25 Antibody, HRP conjugated in research applications.

What are common causes of false results when using TBC1D25 Antibody, HRP conjugated, and how can they be addressed?

When working with TBC1D25 Antibody, HRP conjugated, researchers may encounter several technical challenges that can lead to false results:

• High Background Signal:

  • Causes: Insufficient blocking, excessive antibody concentration, cross-reactivity

  • Solutions: Optimize blocking (3-5% BSA or non-fat milk), increase washing steps, titrate antibody to determine optimal concentration, include appropriate negative controls

• Low or No Signal:

  • Causes: Insufficient target protein, antibody degradation, inhibition of HRP activity

  • Solutions: Confirm target expression in samples, verify antibody activity with positive controls, ensure substrate is fresh and active, check for presence of HRP inhibitors in buffers

• Non-specific Binding:

  • Causes: Cross-reactivity with related proteins, hydrophobic interactions

  • Solutions: Pre-adsorb antibody with related proteins, increase detergent concentration in wash buffers, validate specificity using knockout/knockdown samples

• Variable Results Between Experiments:

  • Causes: Inconsistent conjugation quality, variations in experimental conditions

  • Solutions: Use the same lot of antibody when possible, standardize protocols, include internal controls in each experiment, consider lyophilization to enhance consistency of conjugation

• Reduced HRP Activity:

  • Causes: Exposure to oxidizing agents, extreme pH, sodium azide

  • Solutions: Avoid sodium azide in HRP-related buffers, maintain pH between 6-8, store properly at 4°C with appropriate preservatives

Implementing proper quality control measures and methodically optimizing experimental conditions can minimize these issues and improve reliability of results when working with TBC1D25 Antibody, HRP conjugated.

How can you validate the specificity of TBC1D25 Antibody, HRP conjugated?

Rigorous validation of antibody specificity is essential for reliable research findings. For TBC1D25 Antibody, HRP conjugated, consider these methodological approaches:

• Genetic Models:

  • Test the antibody in samples from TBC1D25 knockout models (as described in cardiac remodeling studies )

  • Compare signal between wild-type and knockout tissues/cells

  • A specific antibody should show signal in wild-type but not in knockout samples

• RNA Interference:

  • Use siRNA or shRNA to knockdown TBC1D25 expression

  • Compare antibody signal before and after knockdown

  • Quantify the reduction in signal relative to the reduction in mRNA

• Recombinant Protein Controls:

  • Test detection of purified recombinant TBC1D25 protein

  • Compare with detection of related TBC domain family proteins

  • Evaluate cross-reactivity and sensitivity

• Peptide Competition:

  • Pre-incubate the antibody with immunizing peptide (1-219AA of human TBC1D25 )

  • A specific antibody will show diminished binding in the presence of the peptide

• Multiple Antibody Validation:

  • Compare results with alternative antibodies against different epitopes of TBC1D25

  • Concordant results with multiple antibodies increases confidence in specificity

• Mass Spectrometry Verification:

  • Immunoprecipitate TBC1D25 and analyze by mass spectrometry

  • Confirm identity of captured proteins matches TBC1D25 sequence

These validation steps ensure that experimental findings accurately reflect TBC1D25 biology rather than artifacts of non-specific antibody binding.

How can TBC1D25 Antibody, HRP conjugated be used in multiplex assays?

Multiplexing allows simultaneous detection of multiple targets, increasing efficiency and providing contextual information about pathway interactions. For TBC1D25 research:

• Multiplex ELISA Approaches:

  • Use spatially separated capture antibodies on segmented plates

  • Combine TBC1D25 (HRP-conjugated) with other antibodies labeled with different enzymes (e.g., alkaline phosphatase) or fluorophores

  • Employ different substrates with distinct detection wavelengths

• Bead-Based Multiplex Assays:

  • Couple capture antibodies to distinct coded beads

  • Detect TBC1D25 binding with HRP-conjugated antibody

  • Simultaneously measure other targets with differently labeled antibodies

  • Sort and analyze using flow cytometry or specialized readers

• Tissue Microarray Analysis:

  • Apply TBC1D25 Antibody, HRP conjugated to tissue microarrays

  • Develop with chromogenic substrates compatible with multiplexing

  • Strip and reprobe, or use spectral unmixing for simultaneous detection of multiple targets

• Proximity Ligation Approaches:

  • Combine TBC1D25 Antibody, HRP conjugated with antibodies against interaction partners

  • Generate signals only when targets are in close proximity

  • Useful for studying TBC1D25-TAK1 interactions in situ

• Sequential Multiplex Immunohistochemistry:

  • Perform initial staining with TBC1D25 Antibody, HRP conjugated

  • Image, then quench HRP activity

  • Repeat with additional antibodies to build a comprehensive profile

These multiplex approaches are particularly valuable for studying the role of TBC1D25 in complex signaling networks such as the TAK1-JNK/p38 pathway in cardiac remodeling .

What therapeutic potential does TBC1D25 research have in cardiac disease?

Research using TBC1D25 Antibody, HRP conjugated has revealed significant therapeutic potential for targeting TBC1D25 in cardiac disease:

• Protective Role in Cardiac Remodeling:

  • TBC1D25 knockout exacerbates cardiac hypertrophy, fibrosis, and dysfunction

  • Overexpression alleviates Angiotensin II-induced cardiomyocyte hypertrophy

  • This suggests TBC1D25 activators or upregulators could protect against pathological cardiac remodeling

• Mechanism-Based Therapeutic Approaches:

  • TBC1D25 suppresses pathological cardiac remodeling by inhibiting TAK1-JNK/p38 signaling

  • Targeting the specific interaction between TBC1D25 and TAK1 (amino acids 138-226 in TBC1D25) could offer precise intervention points

  • Small molecules or peptides that enhance this interaction may have therapeutic value

• Biomarker Development:

  • TBC1D25 is upregulated during pathological cardiac remodeling

  • This suggests potential use as a biomarker for cardiac stress

  • HRP-conjugated antibodies could facilitate development of sensitive diagnostic assays

• Genetic Therapy Approaches:

  • Gene therapy to increase TBC1D25 expression in cardiac tissue

  • CRISPR-based approaches to modify TAK1-binding domains

  • Viral vector delivery of TBC1D25 to cardiac tissue

• Translational Research Pipeline:

  • Pre-clinical models validating TBC1D25 as therapeutic target

  • Development of small molecule modulators of TBC1D25 function

  • Potential clinical applications in heart failure intervention

Research suggests that "TBC1D25 will likely become a promising therapeutic target for heart failure" , highlighting the importance of continued investigation in this area.