TBC1D25 Antibody, FITC conjugated

What is TBC1D25 and why is it significant in autophagy research?

TBC1D25 (TBC1 domain family member 25) functions as a GTPase-activating protein (GAP) specifically targeting RAB33B. Its primary significance lies in regulating autophagosome maturation, where it facilitates the fusion of autophagosomes with endosomes and lysosomes . As a direct binding partner of ATG8 proteins, TBC1D25 represents a critical link between vesicular trafficking and autophagy pathways . This protein contains a functional TBC domain that catalyzes GTP hydrolysis of RAB33B, thereby regulating autophagosome-lysosome fusion events. Understanding TBC1D25's function provides valuable insights into the molecular mechanisms governing autophagy, which has implications for neurodegenerative diseases, cancer, and aging research.

How does the structure of TBC1D25 relate to its function?

TBC1D25 is a 688-amino acid protein with a molecular weight of approximately 76 kDa . Its functional regions include:

• N-terminal region: Contains binding motifs that facilitate interactions with ATG8 family proteins

• TBC domain: Mediates its GAP activity toward RAB33B

• Protein interaction domains: Enable binding to various partners including MAP1LC3A, MAP1LC3B, GABARAPL1, GABARAPL2, GABARAP, FEZ1, and FEZ2

The N-terminal region of TBC1D25 contains the sequence "KVQQVLSWSY GEDVKPFKPP LSDAEFHTYL NHEGQLSRPE ELRLRIYHGG" that serves as an epitope for antibody generation in several commercial antibodies . This region is highly conserved across species, explaining the cross-reactivity of anti-TBC1D25 antibodies with proteins from human, mouse, rat, rabbit, cow, dog, guinea pig, horse, and zebrafish samples .

What controls should be included when using TBC1D25 antibodies in autophagy research?

When designing experiments with FITC-conjugated TBC1D25 antibodies for autophagy studies, the following controls are essential:

Positive controls:

• Cell lines with confirmed TBC1D25 expression (such as HeLa cells in autophagy studies)

• Samples treated with autophagy inducers (starvation, rapamycin) to upregulate autophagy pathways

• Recombinant TBC1D25 protein (1-219AA) can serve as a positive control for antibody specificity

Negative controls:

• TBC1D25 knockout or knockdown cell lines

• Isotype controls (rabbit IgG-FITC) to assess non-specific binding

• Secondary antibody-only controls for indirect detection methods

Treatment controls:

• Bafilomycin A1 (BafA1) treatment to block autophagosome clearance, which should result in increased LC3-II accumulation

• Comparison of steady-state versus starvation-induced autophagy conditions

These controls help validate antibody specificity and ensure that observed signals genuinely represent TBC1D25 in the context of autophagy.

What are the optimal sample preparation methods for TBC1D25 detection?

The optimal sample preparation methods vary by application:

For Western blotting:

• Lyse cells in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS (pH 7.4)

• Include protease inhibitors to prevent protein degradation

• Separate proteins using SDS-PAGE, targeting the ~76 kDa region for TBC1D25

For immunofluorescence:

• Fix cells with 4% paraformaldehyde (10-15 minutes)

• Permeabilize with 0.1-0.5% Triton X-100

• Block with 1-5% BSA or appropriate serum

• Dilute FITC-conjugated TBC1D25 antibody appropriately (1:50-1:200 is recommended for similar antibodies)

For co-localization studies:

• Consider dual labeling with markers for:

  • Autophagosomes (LC3)

  • Lysosomes (LAMP1)

  • Endosomes (EEA1)

  • Retromer complex components (VPS35, VPS29)

How can I resolve weak signal issues when using FITC-conjugated TBC1D25 antibody?

Weak fluorescence signals with FITC-conjugated TBC1D25 antibodies can be addressed through several optimization strategies:

Signal enhancement approaches:

• Adjust antibody concentration (try a titration series from 1:50 to 1:500)

• Extend incubation time (overnight at 4°C can improve signal)

• Use signal amplification systems (tyramide signal amplification)

• Optimize filter sets to match FITC's excitation/emission spectrum

• Adjust exposure settings on imaging equipment

Sample preparation improvements:

• Ensure appropriate fixation (over-fixation can mask epitopes)

• Test different permeabilization agents and concentrations

• Use antigen retrieval methods if necessary

• Consider using stronger blocking agents to reduce background

If signal remains weak, verify TBC1D25 expression levels in your sample using alternative detection methods such as Western blotting with a non-conjugated TBC1D25 antibody.

How do I differentiate between specific binding and autofluorescence when using FITC-conjugated antibodies?

Distinguishing specific FITC-TBC1D25 antibody signals from autofluorescence requires:

Technical approaches:

• Include unstained control samples to assess natural autofluorescence

• Prepare isotype control (rabbit IgG-FITC) samples at the same concentration

• Use spectral unmixing if your microscope has this capability

• Analyze emission spectra (FITC has characteristic emission at ~520 nm)

Validation experiments:

• Perform TBC1D25 knockdown/knockout to confirm signal reduction

• Use multiple antibodies targeting different epitopes of TBC1D25

• Compare localization patterns with published data on TBC1D25 distribution

• Perform colocalization studies with known TBC1D25 interactors such as MAP1LC3B and RAB33B

How can TBC1D25 antibodies be used to study autophagosome maturation dynamics?

FITC-conjugated TBC1D25 antibodies enable sophisticated analysis of autophagosome maturation:

Live cell imaging approaches:

• Transfect cells with mCherry-eGFP-LC3B "traffic light" reporter to track autophagosome-lysosome fusion

• Use FITC-TBC1D25 antibody in fixed time-point experiments to correlate with autophagosome maturation stages

• Analyze colocalization of TBC1D25 with RAB33B and LC3 during autophagy induction

Quantitative analysis methods:

• Measure the ratio of TBC1D25-positive structures that colocalize with early autophagosomes (LC3+/LAMP1-) versus autolysosomes (LC3+/LAMP1+)

• Track changes in TBC1D25 distribution during autophagy induction using high-content imaging

• Perform correlation analysis between TBC1D25 levels and autophagy flux markers

Research has shown that TBC1D25 depletion can substantially reduce BafA1-induced increases in LC3-II during both basal and starvation-induced autophagy, indicating its critical role in autophagosome formation .

What approaches can be used to study TBC1D25's interactions with the retromer complex?

TBC1D25 has been shown to interact with components of the retromer complex, which can be studied using:

Protein interaction analysis:

• Immunoprecipitation followed by Western blotting to detect VPS35, VPS29, and other retromer components

• Proximity ligation assays to visualize TBC1D25-retromer interactions in situ

• FRET/FLIM analysis for studying dynamic interactions

Functional studies:

• Examine the effects of TBC1D25 overexpression or knockdown on retromer localization

• Analyze retromer-dependent trafficking in cells with modified TBC1D25 expression

• Investigate the role of TBC1D25's LIR motifs in mediating interactions with both retromer and autophagy machinery

Research has demonstrated that TBC1D5 (another TBC family member) binds to retromer components through its LIR motifs, and this binding is independent of catalytic activity . Similar mechanisms might apply to TBC1D25, providing a molecular bridge between retromer-mediated trafficking and autophagy.

How can TBC1D25 antibodies be used in quantitative proteomics approaches?

FITC-conjugated TBC1D25 antibodies can be integrated into advanced proteomics workflows:

Immunoprecipitation-mass spectrometry (IP-MS):

• Use TBC1D25 antibodies to immunoprecipitate protein complexes

• Analyze precipitated proteins by LC-MS/MS to identify novel interaction partners

• Compare TBC1D25 interactome under different conditions (basal, starvation, drug treatments)

Quantitative analysis techniques:

• SILAC or TMT labeling to compare TBC1D25-associated proteins across conditions

• Targeted proteomics (MRM/PRM) to quantify specific TBC1D25 interaction partners

• Crosslinking mass spectrometry to identify direct binding interfaces

These approaches have been successfully implemented for other TBC family proteins such as TBC1D5, revealing interactions with retromer components and ATG8 proteins . Similar approaches with TBC1D25 could uncover novel functional connections in autophagy regulation.

How does TBC1D25 conservation across species impact antibody selection?

TBC1D25 exhibits high sequence conservation across multiple species, which has important implications for antibody selection and experimental design:

Cross-reactivity profile:
The available anti-TBC1D25 antibodies show reactivity across numerous species, including:

• Human: 100% sequence recognition

• Mouse: 100% sequence recognition

• Rat: 100% sequence recognition

• Rabbit: 100% sequence recognition

• Cow: 100% sequence recognition

• Dog: 100% sequence recognition

• Guinea pig: 100% sequence recognition

• Horse: 100% sequence recognition

• Zebrafish: 85% sequence recognition

This high degree of conservation allows researchers to use the same antibody across different model organisms, facilitating comparative studies. When designing experiments with non-human samples, it's advisable to validate the antibody in each species of interest, despite the predicted cross-reactivity.