TBC1D25 Antibody

What is TBC1D25 and what cellular functions does it regulate?

TBC1D25 (TBC1 domain family, member 25) is a protein containing a TBC domain that functions as a Rab GTPase activating protein (Rab-GAP). It was previously known as ornithine aminotransferase-like 1 (OATL1), though it has no actual similarity to ornithine aminotransferase . The protein plays a critical role in the fusion of autophagosomes with endosomes and lysosomes, making it an essential component of the autophagy pathway . Recent research has shown that TBC1D25 is also upregulated during pathological cardiac remodeling and appears to suppress cardiac hypertrophy, fibrosis, and dysfunction by regulating the TAK1-JNK/p38 signaling pathway . The protein has an observed molecular weight of approximately 80 kDa and is widely expressed in multiple cell types and tissues across human, mouse, and rat species .

What are the available types of TBC1D25 antibodies and their source organisms?

Based on the search results, there are at least two main types of TBC1D25 antibodies commercially available:

• Monoclonal Antibody (Mouse IgG1):

  • Clone designation: 2C7H3

  • Product number: 67574-1-Ig

  • Host organism: Mouse

  • Format: Unconjugated, liquid form

  • Storage buffer: PBS with 0.02% sodium azide and 50% glycerol (pH 7.3)

• Polyclonal Antibody:

  • Product number: PA5-56082

  • Host organism: Rabbit

  • Format: Unconjugated

  • Immunogen sequence: AVDPQITSLD VLQHILIRAF DLSGKKNFGI SYLGRDRLGQ EVYLSLLSDW DLSTAFATAS KPYLQLRVDI RPSEDSPLLE DWDIISPKDV SGSDVLLAEK RSSLTTAALP FTQSIL

These antibodies are purified using different methods (Protein G purification for the monoclonal antibody and antigen affinity purification for polyclonal antibodies) and show high sequence identity across species, with 96% identity to mouse and 95% to rat orthologs .

What is the reactivity profile of TBC1D25 antibodies across species?

TBC1D25 antibodies show consistent reactivity across multiple mammalian species. The monoclonal antibody (67574-1-Ig) has been tested and confirmed to react with human, mouse, and rat samples . This cross-reactivity is likely due to the high sequence conservation of TBC1D25 across these species. According to the polyclonal antibody information, the immunogen sequence used shows 96% identity to mouse TBC1D25 and 95% identity to rat TBC1D25 .

What are the optimal conditions for using TBC1D25 antibody in Western Blot applications?

For Western Blot (WB) applications using TBC1D25 antibody, the following optimized conditions are recommended:

Dilution Range:

• For monoclonal antibody (67574-1-Ig): 1:1000-1:2000 dilution

Cell Lines Successfully Tested:
The antibody has been validated in multiple cell lines, including:

• NIH/3T3 cells

• HeLa cells

• HEK-293 cells

• Jurkat cells

• HSC-T6 cells

• Neuro-2a cells

Protocol Recommendations:

• Sample preparation: Standard lysis buffers containing protease inhibitors

• Protein loading: 20-30 μg total protein per lane

• Expected band size: 80 kDa (observed molecular weight)

• Blocking: 5% non-fat milk in TBST is typically effective

• Incubation: Primary antibody incubation overnight at 4°C gives optimal results

• Detection: HRP-conjugated secondary antibody followed by ECL detection

For specific optimization needs, the manufacturer provides specialized WB protocols for this antibody that can be downloaded from their website . It's recommended to perform a dilution series during initial optimization to determine the optimal concentration for your specific experimental conditions.

How should TBC1D25 antibody be used for Immunofluorescence and what controls are essential?

For Immunofluorescence (IF) applications with TBC1D25 antibody, the following methodology is recommended:

Dilution Range:

• For monoclonal antibody (67574-1-Ig): 1:400-1:1600 dilution

Validated Cell Lines:
The antibody has been positively tested for IF in:

• NIH/3T3 cells

• HEK-293 cells

Protocol Recommendations:

• Cell fixation: 4% paraformaldehyde (15 minutes at room temperature)

• Permeabilization: 0.1-0.5% Triton X-100 in PBS (10 minutes)

• Blocking: 1-5% BSA or normal serum from the secondary antibody species

• Primary antibody incubation: Overnight at 4°C or 1-2 hours at room temperature

• Secondary antibody: Fluorophore-conjugated secondary antibody matching the host species (mouse for 67574-1-Ig)

• Counterstaining: DAPI for nuclei visualization

• Mounting: Anti-fade mounting medium

Essential Controls:

• Negative control: Omit primary antibody but include all other steps

• Isotype control: Use non-specific mouse IgG1 at the same concentration

• Blocking peptide control: Pre-incubate antibody with blocking peptide

• Knockdown validation: Use cells with TBC1D25 knockdown or knockout

• Subcellular marker co-staining: Co-stain with markers for autophagosomal or endosomal compartments to confirm expected localization

The manufacturer provides specific IF protocols for TBC1D25 antibody that can be downloaded for detailed instructions . Given TBC1D25's role in autophagosome-endosome-lysosome fusion, co-localization studies with markers for these organelles (such as LC3 for autophagosomes or LAMP1 for lysosomes) can provide valuable functional information.

What considerations should be made when using TBC1D25 antibody for immunoprecipitation studies?

While the search results don't specifically mention immunoprecipitation (IP) protocols for TBC1D25 antibody, we can draw on general principles and information from related TBC family antibodies:

Recommended Considerations:

• Antibody Selection:

  • Monoclonal antibodies like 67574-1-Ig may be suitable for IP due to their high specificity

  • The antibody should be validated for IP applications before use

• Protocol Suggestions:

  • Lysate preparation: Use mild lysis buffers (e.g., RIPA buffer with protease inhibitors)

  • Pre-clearing: Pre-clear lysate with Protein G beads to reduce non-specific binding

  • Antibody binding: Incubate 2-5 μg of antibody with 500-1000 μg of total protein lysate

  • Capture: Use Protein G magnetic beads for mouse monoclonal antibodies

  • Incubation time: Overnight at 4°C with gentle rotation

  • Washing: Multiple washes with decreasing salt concentrations

  • Elution: Use gentle elution conditions to preserve protein-protein interactions

• Controls:

  • IgG control: Use non-specific mouse IgG1 as a negative control

  • Input control: Include an aliquot of pre-IP lysate

  • Known interacting partners: Validate by blotting for documented TBC1D25 interactors

• Co-Immunoprecipitation Considerations:

  • Based on research showing TBC1D25 interacts with TAK1, co-IP experiments should be designed to preserve this interaction

  • From search result , TBC1D25 directly interacts with 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

• Validation Methods:

  • Western blot after IP to confirm successful precipitation of TBC1D25

  • Mass spectrometry to identify novel interaction partners

While specific IP protocols for TBC1D25 aren't provided in the search results, these general guidelines combined with the knowledge of TBC1D25's interactions can help design effective IP experiments.

How does TBC1D25 function in the autophagy pathway and what experimental models best demonstrate this?

TBC1D25 plays a crucial role in the autophagy pathway as a Rab GTPase-activating protein (Rab-GAP) that specifically regulates the fusion of autophagosomes with endosomes and lysosomes. This function is critical for the completion of autophagy, as it allows for the degradation of autophagosomal contents .

Molecular Mechanism:
TBC1D25 contains a TBC domain, which is characteristic of Rab-GAPs that inactivate Rab GTPases by stimulating their intrinsic GTPase activity, converting them from the active GTP-bound form to the inactive GDP-bound form. This regulation of Rab GTPases is likely how TBC1D25 controls vesicle fusion events in the autophagy pathway.

Experimental Models to Demonstrate Function:

• Cellular Models:

  • Knockout/Knockdown Systems: TALEN-mediated knockout cells can be generated as described for related TBC family members in search result . This approach allows for clean genetic deletion and functional studies.

  • Overexpression Systems: YFP-tagged or HA-tagged TBC1D25 constructs can be used to study localization and function.

  • Point Mutations: Creating catalytically inactive mutants (similar to the R381A mutation described for TBC1D17) can help determine if the GAP activity is essential for function .

• Autophagy Assays:

  • LC3 Puncta Formation: Monitor autophagosome formation using fluorescently-tagged LC3.

  • Autophagic Flux Assays: Use lysosomal inhibitors (e.g., bafilomycin A1) to assess if TBC1D25 affects autophagosome-lysosome fusion.

  • Long-lived Protein Degradation: Measure the degradation of long-lived proteins as a functional readout of autophagy.

  • Tandem Fluorescent-tagged LC3 (tfLC3): Use mRFP-GFP-LC3 to monitor autophagosome maturation (GFP fluorescence is quenched in acidic lysosomes while RFP remains stable).

• Interaction Studies:

  • Co-localization Analysis: Immunofluorescence studies can demonstrate co-localization with autophagosomal (LC3), endosomal (Rab5, Rab7), and lysosomal (LAMP1, LAMP2) markers.

  • Co-immunoprecipitation: Identify Rab GTPases that interact with TBC1D25.

  • GAP Activity Assays: Measure the GTPase activity of candidate Rab proteins in the presence and absence of TBC1D25.

• In Vivo Models:

  • TBC1D25-KO Mice: As mentioned in search result , TBC1D25-KO mice have been generated and used to study cardiac remodeling. These mice could also be useful for studying the role of TBC1D25 in autophagy in different tissues and under various stress conditions.

These experimental approaches can collectively provide a comprehensive understanding of TBC1D25's function in the autophagy pathway and its potential involvement in related pathological conditions.

What is the role of TBC1D25 in cardiac remodeling and how can researchers study this pathway?

TBC1D25 plays a significant role in cardiac remodeling as revealed by recent research. According to search result , TBC1D25 is upregulated during pathological cardiac remodeling and functions as a protective factor against adverse cardiac remodeling.

Key Findings and Mechanisms:

• Protective Function: TBC1D25 knockout exacerbates cardiac hypertrophy, fibrosis, and dysfunction following partial transverse aortic constriction (TAC), suggesting that TBC1D25 has a cardioprotective role .

• Signaling Pathway Regulation: TBC1D25 suppresses pathological cardiac remodeling by regulating the TAK1-JNK/p38 signaling pathway. Specifically:

  • TBC1D25 deficiency increases phosphorylation levels of TAK1, JNK, and p38

  • Overexpression of TBC1D25 inhibits phosphorylation of these proteins

  • TAK1 appears to be the key molecule in this regulatory process

• Direct Interaction: TBC1D25 directly interacts with TAK1, as demonstrated by immunoprecipitation and GST pull-down assays. This interaction requires:

  • Amino acids 138-226 in the C-terminal region of TBC1D25

  • Amino acids 1-300 in the C-terminal region of TAK1

Experimental Approaches for Studying This Pathway:

• In Vivo Models:

  • TAC Model: Partial transverse aortic constriction in TBC1D25-KO mice and wild-type controls to induce cardiac remodeling

  • Angiotensin II Infusion: An alternative model to induce cardiac hypertrophy and fibrosis

  • Echocardiography: To assess cardiac function in vivo

  • Histological Analysis: For assessment of cardiomyocyte size and cardiac fibrosis

• In Vitro Models:

  • H9C2 Cells and NRCMs (Neonatal Rat Cardiomyocytes): Can be used with TBC1D25 overexpression or knockdown

  • Angiotensin II Treatment: To induce cardiomyocyte hypertrophy in vitro

  • Cell Size Measurement: As a marker of hypertrophy

  • Hypertrophic Gene Expression: Analyze expression of markers like ANP, BNP, and β-MHC

• Molecular Interaction Studies:

  • Co-Immunoprecipitation: To confirm TBC1D25-TAK1 interaction

  • GST Pull-down Assays: To map interaction domains

  • Mutational Analysis: Creating truncated or point mutants to identify critical residues for interaction

  • Proximity Ligation Assay (PLA): To visualize protein-protein interactions in situ

• Signaling Pathway Analysis:

  • Western Blotting: To assess phosphorylation levels of TAK1, JNK, and p38 MAPK

  • Kinase Inhibitors: Use of specific inhibitors (e.g., TAK1 inhibitor 5Z-7-oxozeaenol) to validate pathway involvement

  • Transcriptional Reporters: To measure downstream effects on transcription factors like AP-1

  • RNA-seq/Proteomics: To identify global changes in gene/protein expression

• Translational Relevance:

  • Human Heart Samples: Analysis of TBC1D25 expression in heart failure patients

  • Genetic Association Studies: Examining TBC1D25 polymorphisms in relation to heart disease

  • Therapeutic Targeting: Testing compounds that modulate the TBC1D25-TAK1 interaction

This multi-faceted approach allows researchers to comprehensively study the role of TBC1D25 in cardiac remodeling, from molecular mechanisms to potential therapeutic applications.

How can TBC1D25 antibodies be used to study autophagosome-lysosome fusion events?

TBC1D25 antibodies can serve as valuable tools for investigating autophagosome-lysosome fusion events, given the protein's role in this critical step of the autophagy pathway. Here are methodological approaches for using these antibodies to study fusion dynamics:

1. Co-localization Studies:

• Confocal Microscopy Approach:

  • Use TBC1D25 antibody (67574-1-Ig at 1:400-1:600 dilution) alongside markers for autophagosomes (LC3), late endosomes (Rab7), and lysosomes (LAMP1/2)

  • Quantify co-localization coefficients (Pearson's or Mander's) between TBC1D25 and these markers under various conditions

  • Time-lapse imaging can capture dynamic fusion events with fluorescently-tagged proteins

• Super-resolution Microscopy:

  • Techniques like STORM or STED can provide nanoscale resolution of TBC1D25 localization relative to fusion machinery

  • Particularly useful for resolving the precise spatial arrangement of TBC1D25 at fusion sites

2. Functional Assays:

• Tandem Fluorescent-tagged Reporters:

  • Use mRFP-GFP-LC3 to monitor autophagosome maturation while simultaneously immunostaining for TBC1D25

  • In unfused autophagosomes, both GFP and RFP signals are visible; after fusion with lysosomes, the acidic environment quenches GFP but not RFP

  • Correlation between TBC1D25 presence and fusion events can be quantified

• Lysosomal Enzyme Delivery Assays:

  • Monitor the delivery of lysosomal enzymes to autophagosomes in relation to TBC1D25 expression levels

  • Cathepsin activity within autophagosomes can be measured using activity-based probes

3. Molecular Interaction Analysis:

• Proximity Ligation Assay (PLA):

  • Detect in situ interactions between TBC1D25 and components of the fusion machinery

  • This technique can visualize protein interactions within 40 nm distance in fixed cells

• FRET/BRET Analysis:

  • For live cell studies, combine antibody validation with fluorescent protein-tagged constructs

  • Measure energy transfer between TBC1D25 and fusion machinery components

4. Manipulation Strategies:

• Knockdown/Knockout with Rescue Experiments:

  • Generate TBC1D25-deficient cells using TALEN technology as described in result

  • Rescue with wild-type or mutant TBC1D25 constructs

  • Use antibodies to confirm expression levels in rescue experiments

• Domain-specific Mutations:

  • Create mutations in TBC1D25's Rab-GAP domain to assess functional importance

  • Use antibodies to track localization of these mutants relative to fusion sites

5. Biochemical Fractionation:

• Isolation of Autophagosomes:

  • Perform subcellular fractionation to isolate autophagosomal, endosomal, and lysosomal fractions

  • Use Western blotting with TBC1D25 antibody (67574-1-Ig at 1:1000-1:2000) to determine distribution across fractions

  • Analyze how distribution changes during induction of autophagy or inhibition of fusion

6. Disease-relevant Models:

• Cardiac Hypertrophy Models:

  • Leverage the cardiac protection role of TBC1D25 (from result ) to study how autophagy fusion events may be impacted during cardiac stress

  • Use the TBC1D25 antibody to assess expression changes in response to stress conditions

These methodological approaches provide a comprehensive framework for using TBC1D25 antibodies to study autophagosome-lysosome fusion events in normal physiology and disease states.

What are common issues with TBC1D25 antibody in Western blot and how can they be resolved?

When working with TBC1D25 antibody in Western blot applications, researchers may encounter several common issues. Here are problems that might arise and their recommended solutions:

1. Weak or No Signal:

2. High Background:

3. Multiple Bands or Unexpected Band Size:

4. Optimization Strategies:

5. Sample-specific Considerations:

Based on validation data, the TBC1D25 antibody (67574-1-Ig) works well in multiple cell lines including NIH/3T3, HeLa, HEK-293, Jurkat, HSC-T6, and Neuro-2a cells . If working with other cell types or tissues, additional optimization may be necessary. Consider using one of these validated cell lines as a positive control during troubleshooting.

For detailed, product-specific protocols, the manufacturer provides a downloadable WB protocol specifically for TBC1D25 antibody 67574-1-Ig that may contain additional optimization tips .

How can researchers validate the specificity of TBC1D25 antibody results?

Validating antibody specificity is crucial for ensuring reliable and reproducible research findings. For TBC1D25 antibody, several validation strategies can be employed:

1. Genetic Validation Approaches:

2. Peptide Competition Assay:

• Pre-incubate the antibody with excess immunizing peptide or recombinant TBC1D25 protein

• Apply the neutralized antibody in parallel with regular antibody

• Expected outcome: Significant reduction or elimination of specific signal with neutralized antibody

3. Cross-validation with Multiple Antibodies:

4. Mass Spectrometry Validation:

• Perform immunoprecipitation with TBC1D25 antibody

• Analyze precipitated proteins by mass spectrometry

• Confirm presence of TBC1D25 peptides in precipitated material

5. Orthogonal Methods for Functional Validation:

6. Tissue/Cell Type-Specific Controls:

• Include both positive and negative control samples in experiments

• Positive controls: Cell lines with confirmed TBC1D25 expression (NIH/3T3, HeLa, HEK-293, Jurkat, HSC-T6, Neuro-2a)

• Negative controls: TBC1D25 knockout cells or tissues with minimal expression

7. Recombinant Protein Standards:

• Use purified recombinant TBC1D25 protein at known concentrations

• Create standard curve for quantitative applications

• Verify antibody detection limit and linear range

8. Isotype Control:

• For immunostaining experiments, include isotype control (mouse IgG1 for 67574-1-Ig)

• Process identically to experimental samples

• Helps distinguish specific from non-specific binding

These validation approaches, especially when used in combination, provide robust evidence for antibody specificity and support confidence in experimental results obtained with TBC1D25 antibodies.

How does sample preparation affect TBC1D25 antibody performance in different applications?

Sample preparation has significant effects on antibody performance across different applications. For TBC1D25 antibody, optimizing sample preparation is crucial for obtaining reliable and consistent results:

1. Western Blot Sample Preparation:

2. Immunofluorescence/ICC Sample Preparation:

3. Immunohistochemistry Sample Preparation:

4. Immunoprecipitation Sample Preparation:

5. Critical Factors Affecting Performance Across Applications:

6. Application-Specific Considerations for TBC1D25:

For studying TBC1D25's role in autophagy, sample preparation should account for autophagy dynamics:

• Autophagy induction (starvation, rapamycin) or inhibition (bafilomycin A1) protocols

• Time-course experiments to capture dynamic processes

• Co-immunoprecipitation buffers that preserve interactions with TAK1 and other signaling components

Optimizing sample preparation based on these recommendations will significantly improve the reliability and reproducibility of experiments using TBC1D25 antibodies across different applications.

How can TBC1D25 antibodies be used to investigate its role in disease models?

TBC1D25 antibodies can be valuable tools for investigating the protein's involvement in various disease models, particularly given its roles in autophagy and cardiac remodeling. Here are methodological approaches for different disease contexts:

1. Cardiovascular Diseases:

Based on search result , TBC1D25 plays a protective role against pathological cardiac remodeling, providing a strong foundation for cardiovascular research applications.

2. Neurodegenerative Disorders:

Given TBC1D25's role in autophagy and the importance of autophagy defects in neurodegenerative diseases:

3. Cancer Research:

Autophagy has context-dependent roles in cancer, making TBC1D25 a potential target for investigation:

4. Inflammation and Immunity:

TBC1D25's connection to TAK1 signaling suggests potential roles in inflammation:

5. Methodological Strategies Across Disease Models:

The TBC1D25 antibodies described in the search results (particularly 67574-1-Ig and PA5-56082) have been validated in multiple cell types and applications , making them suitable for these diverse disease research contexts. By applying these antibodies within the suggested experimental frameworks, researchers can gain valuable insights into TBC1D25's contributions to disease pathogenesis and potential therapeutic interventions.

What are the latest research trends involving TBC1D25 and what methodological approaches are being developed?

Based on the search results and emerging research directions, several key trends and methodological innovations are shaping TBC1D25 research:

1. Signaling Pathway Integration:

One of the most significant recent findings is TBC1D25's involvement in the TAK1-JNK/p38 MAPK signaling pathway, particularly in cardiac remodeling . This opens several new research directions:

2. Autophagy Regulation Mechanisms:

TBC1D25's role in autophagosome-endosome-lysosome fusion continues to be an active area of research:

3. Cross-talk Between Autophagy and Inflammation:

The connection between TBC1D25, TAK1 signaling, and autophagy suggests important cross-talk:

4. Advanced Genetic Tools:

The development of precise genetic tools enhances TBC1D25 functional studies:

5. Translational Research Applications:

Moving TBC1D25 research toward clinical applications:

6. Emerging Technology Integration:

These emerging trends and methodological approaches represent the cutting edge of TBC1D25 research, driven by its newly discovered roles in signaling and disease processes. The antibodies described in the search results (67574-1-Ig, PA5-56082) will be essential tools for many of these applications , particularly when integrated with these advanced methodological approaches.