ABHD18 Antibody

What is ABHD18 and why is it relevant to my research?

ABHD18 (abhydrolase domain-containing protein 18) is a member of the AB hydrolase protein superfamily with a canonical length of 414 amino acid residues and a mass of 47 kDa in humans. It has been described as a secreted protein with up to four different isoforms and is widely expressed across many tissue types . Recent research has localized ABHD18 to mitochondria, suggesting potential involvement in cardiolipin regulation .

For research purposes, ABHD18 is particularly interesting because:

• It contains hydrolase domains that may indicate enzymatic activity

• It undergoes post-translational modifications, including glycosylation

• It has been linked to phospholipid metabolism pathways

• It shows evolutionary conservation with orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken species

What applications are ABHD18 antibodies commonly used for?

ABHD18 antibodies are primarily used in several key applications in molecular and cellular biology research:

Some specialized antibodies may also be suitable for immunoprecipitation or flow cytometry, though these applications are less commonly reported for ABHD18 .

How should I select the appropriate ABHD18 antibody for my experiment?

Selection criteria should be based on:

• Target epitope: Consider whether you need an antibody targeting the N-terminal, C-terminal, or internal regions. Different epitopes may be more accessible depending on your application .

• Species reactivity: Ensure the antibody reacts with your species of interest. Available antibodies show reactivity to:

  • Human (Hu)

  • Mouse (Ms)

  • Rabbit (Rb)

  • Rat (Rt)

  • Bovine (Bv)

  • Dog (Dg)

  • Guinea Pig (GP)

  • Horse (Hr)

  • Zebrafish (Zf)

• Application validation: Verify the antibody has been validated for your specific application. Not all antibodies work equally well across different techniques .

• Format: Consider whether you need:

  • Unconjugated antibodies (for most applications)

  • Fluorophore-conjugated (for direct IF or flow cytometry)

  • Enzyme-conjugated (for direct WB, ELISA, or IHC)

How can I validate the specificity of an ABHD18 antibody?

A comprehensive validation approach should include:

• Positive and negative controls:

  • Use tissues/cells known to express or not express ABHD18

  • Include ABHD18 knockout/knockdown samples when available

• Western blot analysis:

  • Confirm a single band (or expected isoform pattern) at the predicted molecular weight (47 kDa for canonical form)

• Orthogonal validation:

  • Compare results with a second antibody targeting a different epitope

  • Correlate protein detection with mRNA expression data

  • Use mass spectrometry validation when possible

• Cross-reactivity testing:

  • Test against related AB hydrolase family members, particularly ABHD2 which may functionally interact with ABHD18

What are optimal protocols for detecting ABHD18 in different subcellular fractions?

ABHD18 has been localized to mitochondria , but is also described as a secreted protein , suggesting multiple cellular locations. For comprehensive detection:

Mitochondrial fraction analysis:

• Isolate pure mitochondrial fractions using differential centrifugation or commercial kits

• Validate fraction purity using markers (e.g., VDAC, COX IV)

• Use standard Western blot protocol with recommended antibody dilutions (1:500-1:1000)

• Include detergent optimization steps (CHAPS, NP-40, or digitonin) to solubilize membrane-associated proteins

• Consider native PAGE for preserving protein complexes

Secreted protein analysis:

• Collect conditioned media from cells

• Concentrate proteins using TCA precipitation or filter concentration

• Conduct Western blot analysis using standard protocols

• Consider glycoprotein-specific detection methods since ABHD18 undergoes glycosylation

How should I optimize immunofluorescence protocols for ABHD18 detection?

For optimal immunofluorescence results:

• Fixation optimization:

  • Test both PFA (4%) and methanol fixation methods

  • For mitochondrial localization, methanol fixation may preserve structure better

• Permeabilization:

  • Use 0.1-0.3% Triton X-100 for general permeabilization

  • For mitochondrial proteins, consider digitonin (0.001-0.01%) for selective membrane permeabilization

• Antibody concentration:

  • Start with the recommended range (0.25-2 μg/mL)

  • Perform a titration to determine optimal signal-to-noise ratio

• Co-localization markers:

  • Include mitochondrial markers (MitoTracker, TOMM20)

  • Consider ER markers to distinguish between secretory pathway and mitochondrial localization

  • Use confocal microscopy for highest resolution analysis

• Signal amplification:

  • Consider tyramide signal amplification for low abundance detection

  • Use high-sensitivity detection systems for weakly expressed isoforms

How can I use ABHD18 antibodies to investigate its potential role in phospholipid metabolism?

Recent research suggests a potential role for ABHD18 in phospholipid metabolism, particularly cardiolipin regulation in conjunction with ABHD2 . To investigate this:

• Co-immunoprecipitation studies:

  • Use ABHD18 antibodies to precipitate protein complexes

  • Analyze precipitated proteins for known phospholipid metabolism enzymes

  • Look specifically for ABHD2 interaction, as these may function together

• Subcellular fractionation with lipidomic analysis:

  • Isolate mitochondria and other cellular fractions

  • Immunoprecipitate ABHD18-containing complexes

  • Perform lipidomic analysis on fractions with and without ABHD18 depletion

  • Focus on cardiolipin species (CL-16.0/18.1/16.0/18.1 has shown strong genetic correlation)

• Activity-based protein profiling:

  • Use activity-based probes designed for hydrolases

  • Confirm binding specificity with ABHD18 antibodies

  • Compare activity profiles between wild-type and ABHD18-depleted samples

• In situ hybridization combined with immunohistochemistry:

  • Correlate mRNA expression with protein localization

  • Analyze co-expression patterns with other phospholipid metabolism enzymes

What are the challenges in detecting different ABHD18 isoforms?

ABHD18 has up to four different reported isoforms , which presents several challenges:

• Isoform-specific detection considerations:

  • Determine which epitopes are present in all isoforms versus isoform-specific regions

  • Consider using antibodies targeting conserved regions for pan-isoform detection

  • For isoform discrimination, use antibodies targeting unique splice junctions

• Experimental approach for isoform characterization:

  • Use high-resolution SDS-PAGE (10-12%) to separate closely sized isoforms

  • Consider 2D gel electrophoresis to separate post-translationally modified variants

  • Validate identities with mass spectrometry after immunoprecipitation

  • Use RT-PCR to correlate protein detection with specific mRNA isoforms

• Tissue-specific expression patterns:

  • Create a systematic tissue expression profile for each isoform

  • Use tissue microarrays with isoform-specific antibodies when available

  • Consider single-cell approaches to detect cell-type-specific expression

• Addressing post-translational modifications:

  • Use glycosidase treatments to assess the impact of glycosylation on detection

  • Consider phosphatase treatments to evaluate phosphorylation states

  • Use specialized mass spectrometry approaches to map all modifications

How can I design experiments to investigate the relationship between ABHD18 and ABHD2?

Given the potential functional relationship between ABHD18 and ABHD2 in regulating cardiolipin levels :

• Co-expression analysis:

  • Use ABHD18 and ABHD2 antibodies to examine co-localization in tissues and cells

  • Quantify correlation of expression levels across tissue panels

  • Analyze temporal expression patterns during development or cellular stress

• Genetic interaction studies:

  • Design CRISPR knockout or knockdown experiments targeting each gene individually and in combination

  • Perform lipidomic analysis focusing on cardiolipin species

  • Consider QTL mapping approaches similar to those that identified the relationship

• Proximity ligation assay (PLA):

  • Use ABHD18 and ABHD2 antibodies from different host species

  • Perform PLA to determine if proteins are within 40nm of each other in situ

  • Quantify interaction signals across different cellular conditions

• Biochemical interaction analysis:

  • Perform sequential immunoprecipitation with both antibodies

  • Use crosslinking approaches to stabilize transient interactions

  • Consider BiFC (Bimolecular Fluorescence Complementation) for in vivo interaction studies

What are common pitfalls when using ABHD18 antibodies and how can I avoid them?

Several challenges may arise when using ABHD18 antibodies:

• Non-specific binding:

  • Problem: Multiple bands on Western blot or diffuse staining in IF/IHC

  • Solution: Optimize blocking conditions (5% BSA often works better than milk for phospho-proteins)

  • Increase washing stringency (higher salt concentration or mild detergents)

  • Pre-absorb antibody with the immunogen peptide as a control

• Mitochondrial localization issues:

  • Problem: Difficulty detecting mitochondrial pools of ABHD18

  • Solution: Use specialized mitochondrial isolation protocols

  • Consider mild permeabilization techniques that preserve mitochondrial integrity

  • Optimize fixation methods (glutaraldehyde/formaldehyde mixtures may better preserve mitochondrial proteins)

• Detection of glycosylated forms:

  • Problem: Heterogeneous banding patterns due to glycosylation

  • Solution: Run control samples treated with PNGase F to remove N-linked glycans

  • Consider using gradient gels to better resolve heterogeneous populations

• Low signal intensity:

  • Problem: Weak or undetectable signal

  • Solution: Try signal amplification methods (HRP polymers, tyramide amplification)

  • Increase antibody concentration or incubation time

  • Consider more sensitive detection systems (ECL-Plus, fluorescent secondary antibodies)

What controls should be included when using ABHD18 antibodies?

A comprehensive control strategy includes:

• Positive controls:

  • Tissues/cells known to express ABHD18 (widely expressed across many tissue types)

  • Recombinant ABHD18 protein where available

  • Overexpression systems (transfected cells)

• Negative controls:

  • Primary antibody omission (to check secondary antibody specificity)

  • Isotype controls (matched concentration of irrelevant antibody)

  • ABHD18 knockout or knockdown samples

  • Pre-immune serum (for polyclonal antibodies)

• Specificity controls:

  • Peptide competition/blocking with immunogen sequence

  • Detection with an independent antibody recognizing a different epitope

  • Correlation with mRNA expression (qPCR or in situ hybridization)

• Technical controls:

  • Loading controls for Western blot (β-actin, GAPDH)

  • Counterstains for morphology in IF/IHC (DAPI, H&E)

  • Subcellular markers to confirm localization (mitochondria: TOMM20, COX IV)

How might ABHD18 antibodies contribute to understanding novel phospholipid regulatory pathways?

ABHD18 antibodies could be instrumental in uncovering new aspects of phospholipid metabolism:

• Mitochondrial membrane dynamics:

  • ABHD18 localization to mitochondria suggests potential roles in membrane organization

  • Use antibodies to track ABHD18 during mitochondrial fission/fusion events

  • Investigate ABHD18 distribution during mitophagy and mitochondrial stress

• Cardiolipin regulation mechanisms:

  • The genetic link between ABHD18 and cardiolipin species suggests regulatory function

  • Study ABHD18 distribution in models of cardiolipin dysregulation

  • Investigate ABHD18 expression changes in mitochondrial diseases

• Integration with other α/β-hydrolase proteins:

  • Many ABHD family members have established roles in lipid metabolism

  • Use antibody panels to map interactions between ABHD proteins

  • Study co-regulation patterns across tissues and disease states

• Potential therapeutic applications:

  • If ABHD18 proves important in cardiolipin metabolism, it may be a target for mitochondrial disorders

  • Antibodies could help validate target engagement of potential ABHD18 inhibitors

  • Consider development of antibody-based detection of ABHD18 as a biomarker

What emerging techniques might enhance ABHD18 antibody applications?

Several cutting-edge approaches could expand ABHD18 antibody utility:

• Proximity labeling proteomics:

  • Fusion of ABHD18 to BioID or APEX2 enzymes

  • Use ABHD18 antibodies to validate proximity labeling results

  • Map the complete ABHD18 interactome in different cellular compartments

• Super-resolution microscopy:

  • Apply STORM, PALM, or STED microscopy using fluorophore-conjugated ABHD18 antibodies

  • Resolve sub-mitochondrial localization patterns

  • Investigate nano-domain organization within membranes

• Single-cell proteomics integration:

  • Combine ABHD18 antibody-based detection with single-cell technologies

  • Map cell-type-specific expression patterns in complex tissues

  • Correlate with single-cell transcriptomics data

• Active learning approaches for antibody-antigen binding:

  • Apply machine learning techniques to optimize ABHD18 antibody binding prediction

  • Use computational approaches to design improved ABHD18-targeting antibodies

  • Develop algorithms to predict cross-reactivity with other ABHD family members