ABHD5 Antibody, Biotin conjugated

What is ABHD5 and what cellular functions does it perform?

ABHD5, also known as CGI-58, is a highly conserved protein that regulates various lipid metabolic pathways through interactions with perilipin (PLIN) and Patatin-like phospholipase domain-containing protein (PNPLA) families . It functions as a coenzyme A-dependent lysophosphatidic acid acyltransferase that catalyzes the transfer of acyl groups on lysophosphatidic acid, contributing to phosphatidic acid biosynthesis . ABHD5 may regulate cellular triacylglycerol storage through activation of the phospholipase PNPLA2 . Additionally, it plays roles in keratinocyte differentiation , lipid droplet fusion regulation , and acts as a tumor suppressor in colorectal cancer by inhibiting YAP-induced c-Met overexpression .

What are the key properties of commercially available ABHD5 antibodies?

Available ABHD5 antibodies exhibit several important characteristics researchers should consider:

What cell lines and tissues are recommended for validating ABHD5 antibodies?

Multiple cell lines have been validated for ABHD5 antibody research, providing reliable positive controls:

When establishing experimental protocols, these validated cell lines serve as appropriate positive controls for assessing antibody performance before proceeding with experimental samples.

What are the recommended dilutions for biotin-conjugated ABHD5 antibodies in different applications?

Optimal dilutions vary by application and specific antibody preparation:

These values serve as starting points; researchers should optimize dilutions for their specific experimental conditions and sample types. Sample-dependent adjustments are often necessary to achieve optimal signal-to-noise ratios .

How should detection protocols be optimized when using biotin-conjugated ABHD5 antibodies?

For optimal detection using biotin-conjugated ABHD5 antibodies, researchers should consider these methodological approaches:

• For immunofluorescence applications, following primary antibody incubation, detection is achieved using avidin-Texas Red conjugate (typically at 1:1000 dilution) .

• In ELISA applications, coat plates with recombinant ABHD5 (100 ng/well) in carbonate/bicarbonate buffer (pH 9.3), incubating overnight at 4°C . Block with PBS containing 0.1% Tween 20 and 5% BSA for 1 hour at 37°C .

• When developing the reaction, biotin-labeled secondary antibodies (typically used at 1:1000 dilution) provide effective detection platforms .

• For multiplex detection, carefully consider fluorophore combinations when using avidin/streptavidin-conjugated reporter molecules to avoid spectral overlap.

What blocking protocols are recommended for minimizing background when using biotin-conjugated antibodies?

Effective blocking is crucial for reducing background signal:

• Use PBS supplemented with 0.1% Tween 20 and 5% BSA as a blocking buffer for ELISA applications .

• Extend blocking time to 1 hour at 37°C to ensure complete coverage of non-specific binding sites .

• For tissue sections or cell preparations with high endogenous biotin (common in liver, kidney), consider implementing biotin/avidin blocking steps before primary antibody application.

• Wash thoroughly (five times with PBS-0.1% Tween 20) following blocking and between antibody incubations .

How can I verify the specificity of ABHD5 antibody binding in my experimental system?

Ensuring antibody specificity requires multiple validation approaches:

• Confirm molecular weight matches the expected 39 kDa in Western blot applications .

• Use paired positive controls (e.g., HepG2, HeLa cells) known to express ABHD5 .

• Include negative controls such as primary antibody omission or isotype controls.

• Consider using both monoclonal and polyclonal antibodies targeting different ABHD5 epitopes for confirmation .

• For critical applications, validate with ABHD5 knockdown or knockout models to confirm signal specificity.

How can biotin-conjugated ABHD5 antibodies be used to study ABHD5-PLIN interactions?

ABHD5-PLIN interactions represent a critical regulatory mechanism in lipid metabolism. Researchers can investigate these interactions using:

• ELISA-based interaction assays with plates coated with 100 ng/well of recombinant ABHD5 .

• Blocking assays to assess anti-PLIN1 autoantibody interference with ABHD5-PLIN1 binding .

• Mapping studies using overlapping peptides to identify specific interaction domains. Research has identified the PLIN1 region 233-405, particularly amino acids 383-405 in the C-terminal domain, as critical for ABHD5 binding .

• Competition experiments using serial dilutions of potentially interfering antibodies (1/250, 1/500, 1/1000) to quantify disruption of protein-protein interactions .

What approaches are recommended for studying ABHD5's role in cancer biology using antibodies?

ABHD5 has emerged as an important tumor suppressor in colorectal cancer through several mechanisms:

• Use ABHD5 antibodies to examine protein expression in cancer vs. normal tissues, particularly in colon cancer models where ABHD5 inhibits YAP-induced c-Met overexpression and cancer stemness .

• Apply co-localization studies with stemness markers (ALDH, CD133, CD44) in cancer cells to investigate correlations between ABHD5 expression and stem cell phenotypes .

• Combine with nuclear/cytoplasmic fractionation approaches to study ABHD5's interaction with DPY30 (a core subunit of the SET1A methyltransferase complex) and its inhibition of DPY30 nuclear translocation .

• Employ animal models (e.g., vil-Cre-pc fl/fl Abhd5 fl/fl mice) to validate in vitro findings regarding ABHD5's role in regulating stemness in tumors .

How can structural analysis inform the selection and application of ABHD5 antibodies?

Recent structural modeling of ABHD5 provides insights for antibody-based research:

• Computational models and topological shape analysis have identified functionally important residues on ABHD5's surface responsible for lipolysis activation by PNPLA2, lipid droplet targeting, and PLIN-binding .

• When selecting antibodies, consider epitopes that target (or avoid) these key functional regions depending on your research question.

• For studying pathological mutations associated with Chanarin-Dorfman Syndrome (CDS), choose antibodies that can still recognize the mutated forms or develop mutation-specific antibodies .

• Use antibody-based approaches to validate computational models by examining the effects of mutating key residues in functional assays .

What are common challenges when using biotin-conjugated ABHD5 antibodies and how can they be addressed?

Several technical issues may arise when working with biotin-conjugated antibodies:

• High Background Signal:

  • Increase washing frequency (five or more washes with PBS-0.1% Tween 20)

  • Optimize blocking conditions (PBS with 0.1% Tween 20 and 5% BSA)

  • Consider endogenous biotin blocking steps, particularly in biotin-rich tissues

• Weak Signal:

  • Adjust antibody concentration within recommended ranges (1:200-1:800 for IF/ICC)

  • Extend incubation times for primary antibody

  • Verify sample preparation and target protein expression

  • Check detection reagent functionality and age

• Inconsistent Results:

  • Standardize protocols for sample preparation

  • Prepare fresh working dilutions for each experiment

  • Include positive control samples (HepG2, HeLa cells)

  • Maintain consistent imaging parameters across experiments

How should ABHD5 localization patterns be interpreted in different experimental contexts?

ABHD5 exhibits dynamic localization patterns that can vary with cellular conditions:

• In basal states, ABHD5 frequently associates with lipid droplets through interactions with perilipin family proteins .

• ABHD5 may shuttle between lipid droplets and other cellular compartments in response to metabolic signals or stimulation .

• In colorectal cancer contexts, ABHD5 has been observed to interact with DPY30 in the cytoplasm, inhibiting DPY30's nuclear translocation and subsequent effects on YAP methylation .

• When comparing immunofluorescence results across experimental conditions, quantify both intensity and subcellular distribution patterns.

• Co-localization with other markers (lipid droplets, nuclear markers, etc.) provides context for interpreting ABHD5's functional state.

What considerations are important when quantifying ABHD5 expression levels using antibody-based approaches?

For accurate quantification:

• Use appropriate housekeeping controls that remain stable under your experimental conditions.

• For Western blot quantification, ensure signal falls within the linear range of detection.

• When comparing ABHD5 expression across different cell types, normalize to relevant parameters and account for differential expression baselines.

• In cancer research contexts, consider that ABHD5 knockdown can significantly increase stemness markers (ALDH, CD133, CD44), providing useful verification points .

• For detecting ABHD5 interactions with partners like PLIN1, titered dilution series in blocking assays can provide quantitative measurements of binding interference .

How might biotin-conjugated ABHD5 antibodies be used to explore emerging roles of ABHD5 in disease pathways?

Emerging research suggests several promising directions:

• Investigate ABHD5's role in methylation regulation through its interaction with DPY30, potentially affecting broader epigenetic patterns beyond YAP methylation .

• Explore ABHD5's function as a tumor suppressor in additional cancer types beyond colorectal cancer.

• Develop multiplex approaches to simultaneously track ABHD5 and its interaction partners (PLIN family proteins, DPY30, SET1A) under various physiological and pathological conditions.

• Apply ABHD5 antibodies in high-throughput screening approaches to identify novel modulators of ABHD5 function as potential therapeutic targets.

• Investigate ABHD5's non-lipid droplet functions and potential roles in other cellular compartments.

What novel technical approaches might enhance ABHD5 antibody applications in research?

Several methodological innovations could advance ABHD5 research:

• Development of proximity-labeling approaches using ABHD5 antibodies to identify novel interaction partners in different cellular contexts.

• Application of super-resolution microscopy techniques to better visualize ABHD5 dynamics relative to subcellular structures.

• Integration of single-cell analysis approaches to examine ABHD5 expression heterogeneity within tissues.

• Combination of antibody-based detection with mass spectrometry to identify post-translational modifications affecting ABHD5 function.

• Development of conformation-specific antibodies that can distinguish between different functional states of ABHD5.