ABHD14B Antibody

What is ABHD14B and what is its primary function in cellular metabolism?

ABHD14B (Abhydrolase Domain Containing 14B) is an enzyme belonging to the metabolic serine hydrolase family. It has been functionally annotated as a lysine deacetylase (KDAC) that transfers an acetyl group from post-translationally modified protein lysine residues to coenzyme A (CoA), generating acetyl-CoA and regenerating the free amine of protein lysine residues .

ABHD14B was initially identified as CCG1/TAF II250-interacting factor B (CIB) through a yeast two-hybrid screen seeking interacting partners of the histone acetyltransferase (HAT) domain of the largest TFIID transcription factor subunit . Recent multi-omics analyses reveal that ABHD14B plays a significant role in regulating glucose metabolism, with its loss resulting in altered glucose metabolism pathways .

Where is ABHD14B predominantly expressed in mammalian tissues?

ABHD14B shows restricted tissue expression patterns. Using selective antibodies against mammalian ABHD14B, tissue distribution surveys in mice have revealed that ABHD14B is predominantly expressed in metabolically active tissues, particularly the liver and kidneys . Additional immunohistochemistry data indicates ABHD14B expression in:

This restricted expression pattern in metabolically active tissues suggests ABHD14B may play an important role in regulating metabolism and cellular energetics .

What is the molecular structure and classification of ABHD14B?

ABHD14B possesses the canonical ABHD fold with an invariant catalytic triad (Ser-His-Asp). The crystal structure of human ABHD14B (PDB: 1IMJ) was determined over a decade ago, revealing that it contains the nucleophilic serine residue (S111) as part of a non-canonical SxxS motif . Based on its protein sequence, ABHD14B is categorized as an outlying member of the metabolic serine hydrolase family, which comprises approximately 1-2% of the total proteome in mammals .

How does ABHD14B function differ from other lysine deacetylases?

ABHD14B represents a novel class of lysine deacetylases distinct from the well-studied sirtuins and histone deacetylase (HDAC) enzymes . While all these enzyme families can deacetylate protein lysine residues, they employ different mechanisms:

This mechanistic distinction makes ABHD14B particularly interesting for researchers studying metabolic regulation, as its activity directly contributes to acetyl-CoA production, a central metabolic intermediate .

What metabolic pathways are affected by ABHD14B knockdown or inhibition?

Transcriptomics and metabolomics analyses in ABHD14B knockdown mammalian cells (HEK293T) reveal that ABHD14B significantly influences glucose metabolism. When ABHD14B is depleted:

• Cellular PKAc levels increase

• Cellular acetyl-CoA concentrations decrease

• Glucose metabolism is altered, with effects on both glycolysis and TCA cycle

• Lactate production is affected, with depleted cellular lactate levels and increased secreted lactate (approximately 2-fold)

Network analysis of differentially expressed genes (DEGs) showed that cellular or primary metabolic processes (particularly glycolysis and the citric acid cycle) were the most overrepresented pathway annotations affected by ABHD14B knockdown .

What experimental approaches are most effective for studying ABHD14B function in vivo?

Given the current lack of specific pharmacological inhibitors and genetic animal models for ABHD14B, researchers have employed several alternative approaches:

• In vivo nonviral transfection: To knockdown hepatic ABHD14B in mice using validated plasmids (e.g., KD_2 and KD_3 plasmids that have demonstrated >95% knockdown efficiency in mammalian cells)

• Multi-omics approach: Combining transcriptomics and metabolomics analyses to comprehensively understand the metabolic consequences of ABHD14B depletion

• Tissue-specific functional studies: Given ABHD14B's restricted expression in metabolically active tissues, targeting liver-specific functions during different metabolic states (e.g., fasting conditions)

• Biochemical assays: Using recombinantly purified human ABHD14B for in vitro deacetylation assays with CoA as substrate

These approaches have revealed that disruption of hepatic ABHD14B, especially during fasting, disturbs homeostatic systemic glucose metabolism and significantly alters organismal energetic status .

What are the optimal dilutions for ABHD14B antibodies in different experimental applications?

Based on validated commercial antibodies, the recommended dilutions vary by application:

For optimal results, it is recommended to titrate the antibody for each testing system, as the optimal dilution may be sample-dependent .

What tissue preparation and antigen retrieval methods are most effective for ABHD14B immunohistochemistry?

For optimal detection of ABHD14B in tissue samples by immunohistochemistry:

• Antigen retrieval recommendations:

  • Primary option: TE buffer pH 9.0

  • Alternative option: Citrate buffer pH 6.0

• Tissue types with validated positive detection:

  • Human prostate hyperplasia tissue

  • Human spleen tissue

  • Mouse small intestine tissue

• Fixation considerations: Standard formalin fixation followed by paraffin embedding (FFPE) is compatible with most commercially available ABHD14B antibodies .

How can researchers validate ABHD14B antibody specificity for their experimental systems?

To ensure antibody specificity and minimize false results:

• Genetic validation:

  • Test antibody response in ABHD14B knockdown systems

  • Compare staining patterns between wildtype and ABHD14B-depleted samples

• Tissue distribution validation:

  • Verify detection primarily in liver and kidney tissues where ABHD14B is known to be highly expressed

  • Confirm low or absent signal in tissues where ABHD14B is not expected

• Western blot molecular weight verification:

  • Confirm detection at expected molecular weight (22-25 kDa)

• Positive and negative controls:

  • Use established positive control samples (L02 cells, mouse small intestine tissue, HepG2 cells)

  • Include biological negative controls (tissues not expressing ABHD14B)

What cell lines are most suitable for ABHD14B functional studies?

Based on research literature, the following cell lines have been successfully used for ABHD14B studies:

For functional studies, HEK293T cells have been effectively used with plasmid-based knockdown approaches (particularly plasmids KD_2 and KD_3) to study the metabolic consequences of ABHD14B depletion .

What controls are essential when designing knockdown experiments for ABHD14B?

For robust ABHD14B knockdown experiments, include:

• Non-targeting (NT) control plasmid: To control for non-specific effects of the transfection procedure

• Untreated "wild-type" cells: To control for potential "off-target" effects of the NT plasmid

• Multiple distinct knockdown constructs: Using two or more different knockdown plasmids (e.g., KD_2 and KD_3) targeting different regions of ABHD14B mRNA helps validate that observed phenotypes are due to ABHD14B depletion rather than off-target effects

• qPCR validation: Confirm knockdown efficiency at the mRNA level

• Western blot validation: Verify protein depletion using a validated ABHD14B antibody

• Rescue experiment: Where possible, include a rescue condition with ABHD14B re-expression to confirm specificity of observed phenotypes

How can researchers distinguish between ABHD14B activity and other deacetylases experimentally?

To specifically attribute deacetylation activity to ABHD14B rather than other cellular deacetylases:

• Cofactor dependency analysis:

  • ABHD14B requires CoA (but not NAD+)

  • Sirtuins require NAD+ (but not CoA)

  • Classical HDACs require neither CoA nor NAD+

• Inhibitor profiling:

  • Use established HDAC inhibitors (e.g., trichostatin A) and sirtuin inhibitors (e.g., nicotinamide) to rule out their contribution

  • ABHD14B activity should be resistant to these inhibitors

• Substrate specificity analysis:

  • ABHD14B shows hydrolase activity towards surrogate p-nitrophenyl substrates with a preference for p-nitrophenyl acetate

  • This distinct substrate profile can help differentiate it from other deacetylases

• Enzymatic assay conditions:

  • Compare deacetylation activity with and without CoA supplementation

  • ABHD14B activity is dependent on CoA as it transfers acetyl groups to CoA

What are the key considerations when preparing tissue samples for ABHD14B immunohistochemical analysis?

For successful detection of ABHD14B in tissue samples:

• Tissue selection: Prioritize liver and kidney tissues where ABHD14B is highly expressed

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Optimization may be necessary for different tissue types

• Antibody dilution titration: Start with manufacturer's recommended range (typically 1:20-1:200) and optimize for your specific tissue samples

• Detection system selection: Both chromogenic and fluorescent detection systems have been successfully used with ABHD14B antibodies

• Controls:

  • Include positive control tissues (liver, kidney, small intestine)

  • Include negative control tissues where ABHD14B is not expressed

  • Include technical negative controls (omitting primary antibody)

• Background reduction: If background is problematic, consider:

  • Additional blocking steps

  • Higher dilution of primary antibody

  • Shorter incubation time

  • Alternative detection systems

How can ABHD14B be targeted for metabolic disease research?

Given ABHD14B's role in glucose metabolism and its restricted expression in metabolically active tissues, several research directions are promising:

• Metabolic syndrome and diabetes: Investigate if ABHD14B dysregulation contributes to impaired glucose homeostasis

• Liver metabolism: Study ABHD14B's role in hepatic glucose metabolism during fasting/feeding cycles

• Acetyl-CoA regulation: Explore how ABHD14B contributes to the cellular acetyl-CoA pool, which affects various metabolic pathways

• Transcriptional regulation: Investigate ABHD14B's potential role in regulating transcription of metabolic genes through its interaction with transcription factors

• Development of specific inhibitors: Design and test small molecule inhibitors of ABHD14B to assess therapeutic potential in metabolic diseases

Research approaches should integrate transcriptomics, metabolomics, and targeted functional studies in both cellular and animal models to fully understand ABHD14B's role in metabolic regulation .

What novel techniques are being developed to study ABHD14B interactions and substrates?

While not explicitly detailed in the available search results, several emerging techniques would be valuable for ABHD14B research:

• Proximity labeling approaches: BioID or APEX2 fusion proteins to identify physiological interaction partners of ABHD14B

• Activity-based protein profiling (ABPP): To identify specific substrates and develop selective inhibitors for ABHD14B

• CRISPR-based genetic models: Development of ABHD14B knockout mice or cell lines for definitive functional studies

• Chemical genetics: Using engineered ABHD14B variants that can be specifically inhibited by bulky inhibitors

• Proteomic identification of acetylation sites: Global acetylome analysis after ABHD14B manipulation to identify its physiological substrates

These approaches would address current gaps in our understanding of ABHD14B biology and potentially reveal new therapeutic targets for metabolic diseases.