ABHD5 Antibody

What is ABHD5 and why is it important in research?

ABHD5 (abhydrolase domain containing 5), also known as CGI-58, is a highly conserved protein that plays crucial roles in lipid metabolism. It functions as an essential coactivator of adipose triglyceride lipase (ATGL), the rate-limiting enzyme in triglyceride hydrolysis. ABHD5 regulates various lipid metabolic pathways through interactions with members of the perilipin (PLIN) and Patatin-like phospholipase domain-containing protein (PNPLA) families .

The significance of ABHD5 extends beyond basic lipid metabolism:

• Loss-of-function mutations result in Chanarin-Dorfman Syndrome (CDS), characterized by ectopic lipid accumulation and severe ichthyosis

• Functions as a tumor suppressor in multiple cancer types including prostate, lung, gastric, liver, and ovarian cancers

• Involved in keratinocyte differentiation and skin barrier formation

• Participates in Hepatitis C Virus assembly and production

ABHD5 antibodies are essential tools for investigating these diverse biological functions in various research settings.

What applications are ABHD5 antibodies validated for?

ABHD5 antibodies have been validated for multiple research applications, with specific commercially available antibodies showing different patterns of reactivity and optimal dilution requirements:

These applications allow researchers to detect and quantify ABHD5 protein levels, localize ABHD5 within cells and tissues, and study protein-protein interactions involving ABHD5 .

What cell lines and tissues show positive ABHD5 antibody reactivity?

Validated ABHD5 antibodies have demonstrated positive reactivity in a wide range of cell lines and tissues:

Cell lines with positive Western blot detection:

• Human cancer cell lines: A431, A549, HCT 116, HeLa, HepG2, LNCaP, Jurkat, K-562, THP-1, Raji, HEK-293

• Mouse cell lines: NIH/3T3

Tissues with positive immunohistochemistry detection:

• Human tissues: colon, kidney, liver, skeletal muscle, small intestine

Cell lines with positive immunofluorescence detection:

• MCF-7 and HepG2 cells

This broad reactivity profile makes ABHD5 antibodies versatile tools for studying this protein across multiple experimental systems and biological contexts .

What are the optimal conditions for using ABHD5 antibodies in Western blotting?

For optimal Western blot results with ABHD5 antibodies, follow these evidence-based recommendations:

Sample preparation:

• ABHD5 is expressed in various tissues with particularly high levels in adipose tissue, liver, and muscle

• Expected molecular weight: 39 kDa (349 amino acids)

• Total protein required: Typically 15-30 μg per lane

Protocol optimization:

• Antibody dilution: Start with 1:1000-1:2000 and optimize based on signal intensity

• Blocking: 5% non-fat milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20)

• Primary antibody incubation: Overnight at 4°C with gentle agitation

• Secondary antibody: Match to host species (rabbit or mouse, depending on primary antibody)

• Detection systems: Both chemiluminescence and fluorescence detection methods are compatible

Antigen retrieval for tissue sections (if applicable):

• TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

The observed molecular weight of 39 kDa corresponds to the calculated molecular weight, suggesting minimal post-translational modifications affect antibody recognition .

How should ABHD5 antibodies be used for immunofluorescence and immunohistochemistry?

For successful immunofluorescence (IF) and immunohistochemistry (IHC) with ABHD5 antibodies:

Immunofluorescence (cells):

• Fixation: 4% paraformaldehyde for 10-15 minutes

• Permeabilization: 0.1-0.5% Triton X-100 for 5-10 minutes

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

• Antibody dilution: 1:200-1:800 (optimal dilution is sample-dependent)

• Counterstaining: DAPI for nuclear visualization

• Expected pattern: Cytoplasmic with potential for punctate signals corresponding to lipid droplets

Immunohistochemistry (tissues):

• Antigen retrieval: TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0

• Blocking: 3-5% serum or protein blocker

• Antibody dilution: 1:500-1:2000

• Detection system: Compatible with DAB (3,3'-diaminobenzidine) visualization systems

• Counterstain: Hematoxylin for nuclear visualization

Positive controls:

• Human tissues: colon, kidney, liver, skeletal muscle

• Cell lines: MCF-7, HepG2 cells

Each antibody should be titrated in the specific testing system to obtain optimal results, as sensitivity may vary between applications and biological samples.

What experimental controls should be included when using ABHD5 antibodies?

Robust experimental design with appropriate controls is essential for reliable results with ABHD5 antibodies:

Positive controls:

• Cell lines with confirmed ABHD5 expression (e.g., HepG2, HeLa, A549)

• Tissues with known ABHD5 expression (e.g., liver, adipose tissue)

• Recombinant ABHD5 protein (where applicable)

Negative controls:

• Primary antibody omission control

• Isotype control (using non-specific IgG of same isotype and concentration)

• ABHD5 knockdown/knockout samples (most definitive control)

• Absorption control (pre-incubating antibody with immunizing peptide)

Validation techniques:

• siRNA knockdown of ABHD5 has been used to confirm antibody specificity

• Some antibodies have been validated using ABHD5 knockout samples

• Comparison of staining patterns between independent antibodies targeting different epitopes

Published literature documents successful ABHD5 knockdown validation in multiple cell lines including Jurkat cells, brown adipocytes, and HCT116 colorectal cancer cells, providing established models for antibody validation .

How can ABHD5 antibodies be used to study protein-protein interactions with ATGL and perilipins?

ABHD5 antibodies enable detailed investigation of protein-protein interactions that regulate lipolysis:

Co-immunoprecipitation (Co-IP) approaches:

• ABHD5 antibodies can be used to immunoprecipitate ABHD5 and detect associated proteins

• For optimal results, use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate

• Protein A/G beads are recommended for rabbit antibodies

• Gentle lysis conditions with non-ionic detergents preserve protein interactions

• Crosslinking may enhance detection of transient interactions

Key ABHD5 interaction partners to analyze:

• ATGL (PNPLA2): Primary target activated by ABHD5

• Perilipin family members (PLIN1, PLIN5): Sequester ABHD5 under basal conditions

• ABHD4: Functionally distinct paralog that diverged from ABHD5 ~500 million years ago

Functional validation approaches:

• Mutagenesis studies have identified key residues (R299 and G328) required for ATGL activation

• ABHD5 mutations R299N and G328S specifically disrupt lipolysis without affecting interactions with perilipin proteins

• Comparative evolutionary analysis between ABHD4 and ABHD5 has revealed structural determinants of lipase activation

These approaches can distinguish between ABHD5's multiple functions: lipid droplet targeting, perilipin binding, and lipase activation.

What are the common challenges in ABHD5 antibody applications and how can they be addressed?

Researchers working with ABHD5 antibodies may encounter several technical challenges:

Challenge 1: Cross-reactivity with related proteins

• ABHD5 belongs to the α/β hydrolase domain family with structural similarity to other members

• Solution: Validate antibody specificity using ABHD5 knockdown/knockout samples

• Alternative: Compare results using antibodies targeting different epitopes

Challenge 2: Limited detection in certain tissues

• ABHD5 expression levels vary significantly across tissues

• Solution: Optimize protein loading (20-40 μg for low-expressing tissues)

• Alternative: Consider enrichment approaches (e.g., immunoprecipitation before Western blot)

Challenge 3: Non-specific background in immunostaining

• May occur due to hydrophobic interactions with lipid-rich structures

• Solution: Increase blocking time/concentration (5% BSA, overnight at 4°C)

• Alternative: Add 0.1-0.3% Triton X-100 to antibody diluent

Challenge 4: Difficulty detecting endogenous protein-protein interactions

• Transient or weak interactions may be difficult to capture

• Solution: Consider in situ proximity ligation assay (PLA) for sensitive detection

• Alternative: Use chemical crosslinking before immunoprecipitation

Challenge 5: Epitope masking in certain conditions

• Protein-protein interactions may shield antibody epitopes

• Solution: Test multiple antibodies targeting different regions of ABHD5

• Alternative: Consider denaturing conditions if detecting total protein is the priority

How can researchers apply ABHD5 antibodies to study its role in cancer progression?

ABHD5 antibodies serve as valuable tools for investigating its tumor suppressor functions:

Research applications in cancer models:

• Immunohistochemical analysis of ABHD5 expression in tumor samples versus normal tissues

• Correlation of ABHD5 protein levels with patient prognosis across cancer types

• Investigation of ABHD5-dependent regulation of mTORC1 signaling through Western blot analysis of phosphorylated p70S6K (Thr389)

• Examination of ABHD5's impact on cell cycle progression using flow cytometry combined with immunofluorescence

Experimental approaches with demonstrated utility:

• ABHD5 overexpression models show G1 cell cycle arrest and growth retardation in prostate cancer cells

• Pharmacological activation of ABHD5-mediated lipolysis inhibits mTORC1 signaling

• ABHD5 expression significantly reduces the ability of lung cancer cells to synthesize nascent proteins

• High ABHD5 expression correlates with extended patient survival in lung, gastric, liver, and ovarian cancers

Mechanistic investigations:

• ABHD5-dependent elevation of intracellular AMP content activates AMPK, leading to inhibition of mTORC1

• This creates an energy-consuming futile cycle between triglyceride hydrolysis and resynthesis

• The process requires DGAT1/DGAT2 isoenzymes that re-esterify fatty acids in an ATP-consuming process

These findings highlight how ABHD5 antibodies can be applied to elucidate novel molecular pathways crucial for limiting cancer cell proliferation.

How do mutations in ABHD5 affect protein function, and how can antibodies help study these effects?

ABHD5 antibodies are instrumental in studying the impact of mutations on protein function:

Key functional mutations identified:

• R299 and G328: Critical for ATGL activation

• E41 and R116: Required for membrane binding

• Y330: Site of binding for NBD-HE-HP affinity ligand

Antibody-based approaches to study mutations:

• Western blotting to assess protein expression levels of mutant proteins

• Immunofluorescence to evaluate subcellular localization changes

• Co-immunoprecipitation to detect altered protein-protein interactions

• Pull-down assays to measure binding to synthetic and endogenous ligands

Disease-relevant mutations:

• Chanarin-Dorfman Syndrome (CDS) mutations can be studied using antibodies to assess:

  • Protein stability and expression levels

  • Subcellular localization changes

  • Altered interactions with functional partners

Experimental design considerations:

• When studying mutants, pair Western blot with functional assays to correlate expression with activity

• For membrane localization studies, combine immunofluorescence with subcellular fractionation

• Use different antibodies targeting distinct epitopes when studying truncation mutants

Research has demonstrated that specific mutations like ABHD5 R299N and G328S selectively disrupt lipolysis without affecting ATGL lipid droplet translocation or ABHD5 interactions with perilipin proteins.

What is known about the structure of ABHD5, and how can antibodies help validate structural models?

Although the crystal structure of ABHD5 remains unsolved, computational models provide valuable insights that can be validated using antibodies:

Current structural understanding:

• ABHD5 belongs to the α/β hydrolase fold family

• Multiple structural models have been developed using homology modeling and advanced deep learning approaches (AlphaFold2)

• The protein contains 8 β sheets and multiple α helices in a canonical alpha/beta hydrolase fold

• Two highly flexible regions: N-terminal (residues 1-52) and insertion helices (residues 198-270)

Antibody-based validation approaches:

• Epitope mapping to confirm predicted surface-exposed regions

• Immunoprecipitation of mutants to validate functional interaction surfaces

• Conformational antibodies that recognize specific protein states

Key structural features identified:

• ABHD5 contains a large binding pocket (pocket α) with a molecular surface area of 1204 Ų and volume of 2586 ų

• Three binding pocket entrances gated by R217, R299, and R116

• Surface patch of Y330 serves as a binding site for the NBD-HE-HP affinity ligand

• Molecular dynamics simulations suggest strong correlations between β strands (β1-β8) that stabilize protein folding

Experimental validation of computationally predicted structures:

• Mutagenesis of predicted functional residues (E41A, R116N, G328E) confirms their importance

• The E41-R116 interaction stabilizes an amphipathic helix critical for membrane binding

These structural insights guide the rational design of experiments to probe ABHD5 function using antibodies directed against specific domains.

How can researchers use ABHD5 antibodies to study ligand binding and pharmacological modulation?

ABHD5 antibodies enable detailed investigation of ligand-protein interactions and drug development efforts:

Approaches to study ligand binding:

• Competitive binding assays: Using labeled probe (e.g., NBD-HE-HP) and measuring displacement by test compounds

• Ligand-induced conformational changes: Detected by altered epitope accessibility

• Pull-down assays: To identify proteins that interact with ABHD5 in ligand-dependent manner

Pharmacological modulators of ABHD5:

• Sulfonyl piperazine (SPZ) compounds disrupt ABHD5 interaction with PLIN1 and PLIN5

• SR-4995 and SR-4559 increase lipolysis ~7-fold in cultured brown adipocytes

• Compound efficacy depends on ABHD5 expression, confirmed through knockdown/rescue experiments

Methodology for binding studies:

• NBD-HE-HP serves as an affinity probe that covalently modifies ABHD5 at Y330

• SR-4995 blocks NBD-HE-HP binding to ABHD5 with similar potency to its disruption of ABHD5-PLIN1 interaction

• Molecular modeling and docking studies suggest hydrophobic interactions are responsible for binding sulfonyl piperazine ligands to ABHD5

Therapeutic implications:

• ABHD5 ligands potentially beneficial for treating metabolic diseases and cancers

• High ABHD5 expression correlates with better prognosis in multiple cancer types

• Activation of ABHD5-mediated lipolysis inhibits mTORC1 signaling and cancer cell growth

Understanding the structural basis of ABHD5-ligand interactions provides critical insights for developing novel therapeutic strategies targeting lipid metabolism disorders and cancer.

How can ABHD5 antibodies be used in studying metabolic disorders beyond Chanarin-Dorfman Syndrome?

ABHD5 antibodies offer valuable tools for investigating various metabolic conditions:

Applications in obesity and insulin resistance research:

• Immunohistochemical analysis of ABHD5 expression in adipose tissue from obese versus lean subjects

• Correlation of ABHD5 protein levels with clinical parameters (BMI, insulin sensitivity)

• Western blot analysis of ABHD5 in response to dietary interventions or exercise

• Evaluation of ABHD5-ATGL interactions during fasting/feeding cycles

Approaches for studying non-alcoholic fatty liver disease (NAFLD):

• Assessment of hepatic ABHD5 expression in different stages of NAFLD/NASH

• Investigation of ABHD5 subcellular localization changes during hepatic steatosis

• Co-localization studies with lipid droplet markers in liver sections

• Analysis of ABHD5-dependent lipolysis regulation in hepatocytes

Methodological considerations:

• For adipose tissue analysis, include perilipin staining to correlate with ABHD5 localization

• In liver samples, consider dual immunofluorescence with hepatocyte markers

• When studying insulin effects, combine with phospho-specific antibodies to assess signaling

• For metabolic flux studies, pair antibody-based detection with functional assays of lipolysis

Clinical relevance:

• ABHD5 activation may represent a therapeutic approach for metabolic diseases

• Monitoring ABHD5 protein levels could serve as a biomarker for metabolic health

• ABHD5-ATGL axis provides a link between lipolysis dysfunction and disease progression

These applications extend the utility of ABHD5 antibodies beyond basic research into clinically relevant investigations of metabolic regulation.

What is the current understanding of post-translational modifications of ABHD5 and how can antibodies detect these changes?

Research into ABHD5 post-translational modifications (PTMs) represents a growing area where specialized antibodies can provide valuable insights:

Current knowledge of ABHD5 PTMs:

• The calculated molecular weight (39 kDa) generally matches observed migration on SDS-PAGE

• This suggests minimal impact of PTMs on protein migration behavior

• Limited research exists on specific PTMs of ABHD5, representing a knowledge gap

• Potential phosphorylation sites have been predicted but require experimental validation

Antibody-based approaches to study PTMs:

• Phospho-specific antibodies: Could be developed against predicted phosphorylation sites

• Mobility shift assays: Using standard ABHD5 antibodies to detect PTM-induced migration changes

• Two-dimensional gel electrophoresis: Combined with Western blot to resolve PTM variants

• Immunoprecipitation followed by mass spectrometry: To identify unknown modifications

Experimental considerations:

• Include phosphatase treatment controls when investigating phosphorylation

• Consider analysis under different metabolic conditions (fasting, insulin stimulation)

• Examine tissue-specific regulation of PTMs

• Evaluate the impact of PTMs on protein-protein interactions and enzymatic activity

Future research directions:

• Development of modification-specific antibodies when key PTM sites are confirmed

• Investigation of how PTMs affect ABHD5's ability to activate ATGL

• Analysis of PTM changes in disease states

• Exploration of PTM-mediated regulation of ABHD5 subcellular localization

This emerging area offers significant opportunities for novel antibody development and application in understanding ABHD5 regulation at the post-translational level.

How can researchers apply ABHD5 antibodies in high-throughput and single-cell analysis technologies?

Emerging technologies offer exciting opportunities to apply ABHD5 antibodies in novel research contexts:

Applications in high-throughput screening:

• Automated immunofluorescence for compound library screening against ABHD5-PLIN interactions

• Cell-based assays measuring ABHD5 translocation in response to drug candidates

• High-content imaging to simultaneously evaluate ABHD5 localization and metabolic parameters

• Tissue microarray analysis of ABHD5 expression across large patient cohorts

Single-cell analysis approaches:

• Mass cytometry (CyTOF) incorporating ABHD5 antibodies for multi-parameter single-cell analysis

• Single-cell Western blotting to detect ABHD5 in rare cell populations

• Imaging mass cytometry for spatial resolution of ABHD5 in complex tissues

• In situ proximity ligation assay for visualizing ABHD5-protein interactions at single-cell resolution

Technical considerations:

• Antibody validation is especially critical for high-throughput applications

• For single-cell approaches, confirm antibody performance at lower protein concentrations

• Consider dual antibody approaches targeting different epitopes for increased specificity

• Appropriate positive and negative controls are essential for reliable data interpretation

Research applications:

• Heterogeneity analysis of ABHD5 expression in tumor microenvironments

• Cell-type specific roles of ABHD5 in metabolic tissues

• Dynamic changes in ABHD5-protein interactions during cellular processes

• Correlation of ABHD5 levels with single-cell metabolic profiles

These advanced applications extend the utility of ABHD5 antibodies beyond traditional biochemical assays into the realm of systems biology and precision medicine.