VPS18 Antibody, Biotin conjugated

What is VPS18 and what cellular functions does it serve?

VPS18 (Vacuolar protein sorting-associated protein 18 homolog) is a critical component in vesicle-mediated protein trafficking to lysosomal compartments, functioning in both endocytic membrane transport and autophagic pathways. It serves as a core component of the putative HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering) endosomal tethering complexes . These complexes are involved in the Rab5-to-Rab7 endosome conversion process and mediate tethering and docking events during SNARE-mediated membrane fusion . VPS18 is essential for the fusion of endosomes and autophagosomes with lysosomes, making it a critical protein for cellular degradative processes . Additionally, immunoprecipitation experiments have shown that mammalian VPS18 (mVps18) interacts with multiple Syntaxin proteins (Syn7, vesicle-associated membrane protein 8, Vti1-b, Syn13, and Syn6) as well as the Sec1/Munc18 protein mVps45, which collectively catalyze early endosomal fusion events .

How do biotin-conjugated antibodies enhance protein detection assays?

Biotin-conjugated antibodies represent a powerful tool in protein detection due to the extremely high affinity between biotin and streptavidin. This conjugation significantly enhances detection sensitivity through signal amplification capabilities. When a biotin-conjugated antibody binds to its target protein, the subsequent addition of a streptavidin-enzyme complex (typically streptavidin-HRP or streptavidin-AP) provides strong signal amplification . The biotin-streptavidin interaction is one of the strongest non-covalent interactions in biology, making it highly stable during washing steps and reducing background noise. In research applications, biotin-conjugated antibodies are particularly valuable for detecting low-abundance proteins like transcription factors or proteins in limited sample quantities . Additionally, the small size of biotin molecules minimizes steric hindrance that might otherwise interfere with antibody-antigen binding, maintaining the native specificity of the unconjugated antibody.

What experimental applications benefit most from biotin-conjugated VPS18 antibodies?

Biotin-conjugated VPS18 antibodies are particularly valuable in several experimental applications:

ELISA and Western Blotting: The biotin-streptavidin amplification system provides enhanced sensitivity for detecting VPS18 in complex protein mixtures. This is especially important when studying low-abundance proteins in the endosomal trafficking pathway .

Immunoprecipitation: Biotin-conjugated VPS18 antibodies excel in immunoprecipitation studies, allowing researchers to effectively isolate VPS18 and its interaction partners. Based on published data, VPS18 antibodies have successfully immunoprecipitated the protein from HeLa cells .

Immunohistochemistry and Immunofluorescence: For tissue-based detection, biotin-conjugated antibodies provide superior signal-to-noise ratios. VPS18 antibodies have been successfully used in IHC applications on human lung cancer tissue, human testis tissue, mouse brain tissue, and mouse testis tissue .

Co-localization Studies: When studying VPS18's association with endosomal compartments or other proteins in the endocytic pathway, biotin-conjugated antibodies allow for flexible experimental design through compatibility with multiple detection systems .

What are optimal protocols for using biotin-conjugated VPS18 antibodies in Western blotting?

When using biotin-conjugated VPS18 antibodies for Western blotting, consider the following optimized protocol:

• Sample Preparation: Prepare cell or tissue lysates with a complete protease inhibitor cocktail, as VPS18 may be subject to degradation. For brain tissue samples (where VPS18 is expressed), use cooled RIPA buffer with phosphatase inhibitors .

• Gel Selection: Use 8-10% SDS-PAGE gels due to VPS18's relatively high molecular weight (observed at 100-110 kDa) .

• Protein Transfer: Perform transfer to PVDF membranes at lower current for longer duration (overnight at 30V) to ensure complete transfer of high molecular weight proteins.

• Blocking: Block membranes with 5% non-fat dry milk in TBST buffer (PBS with 0.02% sodium azide and 50% glycerol pH 7.3 is recommended for storage of the antibody itself) .

• Antibody Dilution: Use a dilution range of 1:500-1:1000 for primary biotin-conjugated VPS18 antibody, incubating overnight at 4°C .

• Detection System: Apply streptavidin-HRP (1:5000-1:10000) for 1 hour at room temperature.

• Development: Develop using enhanced chemiluminescence, with exposure times optimized based on signal strength.

• Controls: Include positive control samples such as HeLa cell lysate, which has been validated for VPS18 detection .

How should I approach antigen retrieval for VPS18 immunohistochemistry?

Effective antigen retrieval is critical for successful VPS18 immunohistochemistry. Based on published protocols:

• Heat-Induced Epitope Retrieval (HIER): The recommended approach uses TE buffer at pH 9.0, which has proven effective for VPS18 detection in multiple tissue types including human lung cancer tissue, human testis tissue, mouse brain tissue, and mouse testis tissue .

• Alternative Method: Citrate buffer at pH 6.0 can serve as an alternative, though potentially with reduced epitope exposure .

• Protocol Details:

  • Deparaffinize and rehydrate tissue sections completely

  • Immerse slides in preheated retrieval buffer

  • Maintain at 95-98°C for 15-20 minutes

  • Allow to cool gradually at room temperature for 20 minutes

  • Wash thoroughly in PBS before proceeding to blocking

• Antibody Dilution: Use a dilution range of 1:50-1:500 for IHC applications, with specific optimization recommended for each tissue type .

• Detection System: For biotin-conjugated antibodies, employ streptavidin-HRP or streptavidin-AP systems, followed by appropriate chromogenic substrate development.

How can I determine optimal dilution factors for biotin-conjugated VPS18 antibodies?

Determining the optimal dilution for biotin-conjugated VPS18 antibodies requires systematic titration across different applications:

• Titration Approach: Conduct a series of dilutions spanning the recommended range (1:500-1:1000 for WB, 1:50-1:500 for IHC, 1:200-1:800 for IF/ICC) .

• Application-Specific Considerations:

  • For Western blotting: Start with 1:500, 1:750, and 1:1000 dilutions using identical samples

  • For IHC: Begin with broader dilution ranges (1:50, 1:200, 1:500) on control tissues

  • For IF/ICC: Test 1:200, 1:400, and 1:800 dilutions on A549 cells, which have been validated for VPS18 detection

• Signal-to-Noise Assessment: Evaluate not only signal intensity but also background levels. The optimal dilution provides maximum specific signal while minimizing non-specific background.

• Positive Controls: Include samples known to express VPS18, such as HeLa cells for IP or human lung cancer tissue for IHC .

• Quantitative Analysis: When possible, perform densitometric analysis of Western blots or quantitative image analysis of IHC/IF samples to objectively determine optimal signal-to-noise ratios.

• Validation: Confirm findings with complementary detection methods or alternative antibodies against VPS18 to ensure consistency.

How can biotin-conjugated VPS18 antibodies be used to study endosomal trafficking dynamics?

Researching endosomal trafficking dynamics with biotin-conjugated VPS18 antibodies can provide valuable insights through multiple experimental approaches:

• Co-localization Studies: Design dual-labeling experiments using biotin-conjugated VPS18 antibodies alongside markers for different endosomal compartments. For early endosomes, use markers like early endosome antigen-1 (EEA1) and transferrin receptor, which have been shown to co-localize with mammalian VPS components in Vero, normal rat kidney, and Chinese hamster ovary cells . For late endosomes/lysosomes, use LAMP1/2 markers to establish VPS18's role in the endocytic pathway.

• Live-Cell Dynamics: Combine immunostaining of fixed cells at different time points after endocytosis induction with pulse-chase experiments using fluorescently-labeled cargo proteins to analyze the temporal relationship between VPS18 and endocytic progression.

• Interaction Partners Analysis: Use biotin-conjugated VPS18 antibodies for co-immunoprecipitation studies to identify and quantify interactions with proteins like Syntaxin 7, vesicle-associated membrane protein 8, and Vti1-b, which have been shown to interact with mVps18 . These interactions can be studied under different cellular conditions to understand regulatory mechanisms.

• Cytoskeletal Connections: Investigate VPS18's association with cytoskeletal elements, as research has shown that mVps18, mVps16, and mVps11 associate with actin filaments, which may coordinate their activity in endosomal trafficking .

• Functional Knockdown Correlation: Combine antibody-based localization studies with siRNA knockdown of VPS18 to correlate localization patterns with functional defects in endosomal trafficking.

What approaches effectively validate VPS18 antibody specificity in experimental systems?

Thorough validation of VPS18 antibody specificity is essential for generating reliable research results. Implement these complementary validation approaches:

• Western Blot Analysis: Confirm that the antibody detects a single band of appropriate molecular weight (100-110 kDa for VPS18) in positive control samples like HeLa cells or mouse brain tissue. Compare multiple tissue/cell types with known differential expression patterns.

• Knockout/Knockdown Controls: Apply the antibody in VPS18 knockout or knockdown models (CRISPR/Cas9 or siRNA) to confirm the absence or reduction of signal. This represents the gold standard for antibody validation.

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (such as the synthetic peptide within Human VPS18 aa 1-50 used for ab240574) before application to your sample. A specific antibody will show diminished or absent signal when pre-blocked with its cognate peptide.

• Recombinant Protein Controls: Test the antibody against purified recombinant VPS18 protein, such as GST or MBP fusion proteins of VPS18 domains , to verify direct recognition.

• Orthogonal Method Comparison: Compare protein detection patterns using alternative methods like mass spectrometry or RNA expression (qPCR) to correlate protein levels with antibody signal intensity.

• Cross-Reactivity Assessment: Test the antibody against closely related proteins (other HOPS complex components) to ensure it doesn't cross-react with structural homologs.

• Multiple Antibody Comparison: When possible, compare detection patterns with multiple antibodies targeting different epitopes of VPS18.

How can I design experiments to investigate VPS18's role in the HOPS and CORVET complexes?

Investigating VPS18's function within HOPS and CORVET complexes requires sophisticated experimental approaches:

• Complex Component Co-IP: Utilize biotin-conjugated VPS18 antibodies for co-immunoprecipitation studies to pull down intact HOPS or CORVET complexes. Analyze precipitated proteins by mass spectrometry or Western blotting for known complex components to determine complex integrity and stoichiometry under different cellular conditions .

• Sequential IP Strategy: Design sequential immunoprecipitation experiments using antibodies against different complex components followed by VPS18 antibodies to distinguish between HOPS-associated and CORVET-associated VPS18 pools.

• Functional Reconstitution: Combine purified components of HOPS/CORVET complexes with biotin-conjugated VPS18 antibodies in in vitro fusion assays to determine the specific role of VPS18 in membrane tethering and fusion events.

• Rab Conversion Studies: Design experiments to investigate VPS18's role in Rab5-to-Rab7 conversion by immunoprecipitating VPS18 at different stages of endosome maturation and analyzing associated Rab proteins .

• Structural Interaction Mapping: Use biotin-conjugated antibodies against specific domains of VPS18 to map the interactions with other HOPS/CORVET components and determine which domains are critical for complex formation and function.

• Subcellular Fractionation: Combine with density gradient fractionation to isolate endosomal compartments at different maturation stages, then analyze VPS18 distribution and complex formation across fractions.

How can I address common challenges when using biotin-conjugated VPS18 antibodies in immunoprecipitation?

Immunoprecipitation with biotin-conjugated VPS18 antibodies may present several challenges that can be systematically addressed:

• Non-specific Binding: To reduce background, use more stringent washing buffers (increasing salt concentration from 150mM to 250-300mM NaCl) and include 0.1-0.5% non-ionic detergents. Pre-clear lysates with unconjugated beads before adding the antibody.

• Weak Signal: For VPS18 IP, use the recommended 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . Consider increasing antibody concentration or incubation time (overnight at 4°C) for improved capture efficiency.

• Interfering Factors: When using streptavidin beads with biotin-conjugated antibodies, endogenous biotin in some cell types may interfere. Pre-clear lysates with streptavidin beads before adding biotin-conjugated antibodies.

• Complex Stability: VPS18 functions within protein complexes (HOPS and CORVET) , which may be sensitive to extraction conditions. Use gentler lysis buffers (with lower detergent concentrations) and avoid harsh detergents like SDS to maintain complex integrity.

• Co-IP Partner Detection: When studying VPS18 interaction partners like Syntaxins and other SNARE proteins , optimize Western blotting conditions specifically for these partners, which may have different detection requirements than VPS18 itself.

• Antibody Cross-Reactivity: Validate the specificity of the biotin-conjugated VPS18 antibody by performing IPs from VPS18-knockdown cells as negative controls to ensure signals are specific.

• Elution Conditions: For biotin-conjugated antibodies, avoid harsh elution conditions that might denature co-precipitated proteins. Consider non-denaturing elution with excess biotin or specific peptide competition.

What fixation methods optimize VPS18 epitope preservation in immunofluorescence studies?

The choice of fixation method significantly impacts VPS18 detection in immunofluorescence applications:

• Paraformaldehyde Fixation: 4% PFA for 15-20 minutes at room temperature generally preserves VPS18 epitopes while maintaining cellular architecture. This is particularly important when studying the localization of VPS18 in relation to endosomal compartments .

• Methanol Fixation Considerations: Cold methanol fixation (-20°C for 10 minutes) may better preserve some epitopes while simultaneously permeabilizing cells, but can disrupt membrane structures relevant to VPS18's function in vesicular trafficking.

• Hybrid Approaches: A combination of 2% PFA (10 minutes) followed by methanol treatment can preserve both protein epitopes and subcellular structures for co-localization studies.

• Permeabilization Optimization: When using PFA fixation, test different permeabilization agents (0.1-0.5% Triton X-100, 0.05-0.2% saponin, or 0.05% digitonin) to find the optimal balance between epitope accessibility and structural preservation.

• Validation Approach: Compare multiple fixation methods side-by-side on the same cell type (A549 cells have been validated for VPS18 immunofluorescence) to determine which method yields the strongest specific signal with minimal background.

• Antigen Retrieval Adaptation: For challenging samples, adapt antigen retrieval methods from IHC protocols, such as using citrate buffer (pH 6.0) or TE buffer (pH 9.0) in a controlled heating step prior to antibody application .

• Post-Fixation Storage: If fixed samples cannot be processed immediately, store in PBS with 0.02% sodium azide at 4°C for short-term storage to preserve epitope integrity .

How should I interpret VPS18 expression patterns across different tissue types?

Interpreting VPS18 expression patterns requires careful consideration of tissue-specific contexts and experimental variables:

• Baseline Expression Patterns: VPS18 has been detected in multiple tissues including human lung cancer tissue, human testis tissue, mouse brain tissue, and mouse testis tissue . When analyzing new tissue types, compare expression levels to these validated positive controls.

• Subcellular Localization Analysis: In normal cells, VPS18 shows significant localization to early endosome antigen-1 (EEA1) and transferrin receptor-positive early endosomes in multiple cell types including Vero, normal rat kidney, and Chinese hamster ovary cells . Deviations from this pattern may indicate altered endosomal trafficking.

• Quantitative Approach: Implement quantitative image analysis to measure VPS18 signal intensity across different cellular compartments, establishing a numeric baseline for normal expression patterns that can be compared across experimental conditions.

• Correlation with Function: Interpret VPS18 expression in the context of endosomal trafficking efficiency. Changes in expression may correlate with alterations in protein degradation pathways, autophagy, or lysosomal function .

• Pathological Considerations: In diseased tissues (like cancer samples), changes in VPS18 expression or localization may reflect broader alterations in vesicle trafficking pathways. Compare against matched normal tissues when possible.

• Development Context: Consider developmental stage when interpreting VPS18 expression, as it is involved in dendrite development of Purkinje cells and may show stage-specific expression patterns .

• Technical Considerations: Account for variables such as antibody concentration, detection method sensitivity, and tissue preparation when comparing expression levels across different experiments or publications.

What are the critical considerations when analyzing VPS18's interactions with the cytoskeleton?

VPS18's association with cytoskeletal elements represents an important aspect of its function in coordinating vesicular trafficking:

• Differential Cytoskeletal Associations: Research has shown that mVps18, mVps16, and mVps11 associate primarily with actin filaments, while related proteins mVam2 and mVam6 associate with microtubules . When designing co-localization experiments, include markers for both cytoskeletal systems.

• Functional Significance: Interpret cytoskeletal associations in the context of vesicle movement mechanisms. Actin-associated VPS18 may function differently than microtubule-associated populations in terms of endosomal positioning and movement.

• Hook1 Interaction Analysis: VPS18 interacts with the microtubule-associated Hook1 protein , suggesting complex interactions across cytoskeletal systems. Include Hook1 in co-immunoprecipitation studies with biotin-conjugated VPS18 antibodies.

• Dynamic vs. Static Associations: Consider whether VPS18-cytoskeleton interactions are stable or transient. Time-course experiments following vesicle trafficking may reveal temporal patterns in these associations.

• Experimental Approach Integration:

  • Combine biochemical assays (co-sedimentation, in vitro binding) with imaging studies

  • Correlate antibody-based detection with live-cell imaging using fluorescently tagged VPS18

  • Use cytoskeleton-disrupting agents (cytochalasin D for actin, nocodazole for microtubules) to functionally test the relevance of these interactions

• Compartment-Specific Analysis: Determine whether VPS18-cytoskeleton interactions differ between early endosomes, late endosomes, and lysosomes, potentially reflecting stage-specific trafficking requirements.

• Quantification Methods: Implement rigorous quantification approaches such as Pearson's correlation coefficient for co-localization analysis and intensity correlation analysis for determining the strength of associations.