ALKBH4 Antibody Biotin conjugated

ALKBH4 Antibody: Technical Overview

Target Protein: ALKBH4 (AlkB homolog 4, lysine demethylase), encoded by the ALKBH4 gene, is a member of the ALKB family of non-heme Fe(II)/α-ketoglutarate (α-KG)-dependent dioxygenases. ALKBH4 functions primarily in the oxidative demethylation of lysine residues, with emerging roles in transcriptional regulation, actin cytoskeleton modulation, and cell cycle control.

Species Reactivity: Most ALKBH4 antibodies are designed to react with human ALKBH4; cross-reactivity has been reported in Mus musculus and Rattus norvegicus, depending on sequence homology at the epitope region.

Immunogen: The immunogen is typically a synthetic peptide corresponding to a region within the conserved C-terminal domain or a full-length recombinant human ALKBH4 protein. Selection of the immunogenic region often excludes the catalytic DSBH (double-stranded β-helix) fold to increase specificity for non-catalytic conformational epitopes.

Clonality: Available in both monoclonal and polyclonal formats:

• Monoclonal antibodies offer batch-to-batch consistency, high specificity, and are ideal for IHC and quantitative assays.
• Polyclonal antibodies provide broader epitope coverage and higher signal in western blot (WB) or IP, though with less reproducibility.

Host Species: Commonly raised in rabbit (monoclonals via recombinant rabbit hybridoma or phage display platforms), goat, or mouse.

Applications and Validation

• Western Blot (WB): Detects ALKBH4 at ~28–35 kDa, depending on post-translational modifications or splice variants. SDS-PAGE under reducing conditions is typically required for optimal band resolution.
• Immunocytochemistry (ICC) / Immunofluorescence (IF): Localizes ALKBH4 predominantly in the nucleus and cytoplasm. Proper fixation (e.g., 4% paraformaldehyde) and permeabilization protocols (e.g., Triton X-100) are critical for preserving subcellular architecture.
• Immunohistochemistry (IHC-P): Paraffin-embedded tissue sections have shown ALKBH4 expression in epithelial tissues, notably in gastrointestinal and reproductive tract epithelium.
• Chromatin Immunoprecipitation (ChIP): Antibodies that recognize DNA-bound ALKBH4 are utilized to map chromatin association profiles, implicating ALKBH4 in transcriptional control networks.
• Co-Immunoprecipitation (Co-IP): Used to identify ALKBH4 interacting partners, including cytoskeletal regulators and chromatin-associated factors.

Pathophysiological Relevance

• Actin Cytoskeleton Regulation: ALKBH4 interacts with actin-binding proteins and modulates cytoskeletal dynamics via hydroxylation of lysine residues on non-histone proteins. Disruption of ALKBH4 leads to defects in cytokinesis and cell polarity.
• Cancer Biology: Downregulation or mislocalization of ALKBH4 has been linked to tumorigenesis in colorectal and prostate cancers, potentially via dysregulated epigenetic modification and cytoskeletal destabilization.
• Epigenetic Modulation: While not a canonical histone demethylase, ALKBH4 exhibits putative demethylase activity on mono- or di-methylated lysine residues, contributing to non-histone lysine methylation dynamics.

Storage and Handling

• Formulation: Typically provided in Tris or PBS buffer with 0.02–0.05% sodium azide and 50% glycerol for stability. Lyophilized forms should be reconstituted in sterile water or assay buffer per datasheet instructions.
• Storage: -20°C for long-term storage; repeated freeze-thaw cycles should be avoided. For frequent use, aliquoting is recommended.
• Concentration: Varies by lot, generally in the range of 0.2–1.0 mg/mL.

Epitope Mapping and Specificity Controls

• Epitope validation using peptide competition assays and ALKBH4 knockout or knockdown cell lines (e.g., CRISPR-Cas9 edited HEK293T or HCT116) confirms specificity.
• Sequence alignment across ALKBH family members is essential to assess cross-reactivity, especially with structurally related paralogs like ALKBH3 and ALKBH5.