H2AV Antibody

H2AV Antibody Overview

H2AV antibodies specifically target the Drosophila histone variant H2AV, a chimera of H2AZ (involved in chromatin stability) and H2AX (critical for DNA repair). These antibodies detect either the unmodified H2AV protein or its phosphorylated form (γ-H2AV), which is a marker of DNA double-strand breaks (DSBs) .

Key Applications

• Western Blot (WB): Detects H2AV (~14 kDa) or γ-H2AV in tissue lysates .

• Immunofluorescence (IF): Visualizes H2AV localization in Drosophila tissues, such as lymph glands or oocytes .

• Chromatin Immunoprecipitation (ChIP): Maps H2AV incorporation into chromatin .

• ELISA: Quantifies H2AV phosphorylation levels in response to DNA damage .

Immune Regulation

• Loss of H2AV enhances immune response:
  Knockdown of H2Av via RNAi in Drosophila fat bodies or S2 cells upregulates antimicrobial peptides (AMPs) like Attacin, Cecropin, and Diptericin through the IMD pathway. This suggests H2Av acts as a negative regulator of innate immunity .

  • Mechanism: H2Av depletion disrupts chromatin structure, potentially derepressing AMP genes .

Hematopoiesis and Melanotic Tumor Formation

• H2AV maintains hematopoietic homeostasis:
  Homozygous His2Av mutants (e.g., His2Av 810) develop melanotic tumors due to premature differentiation of prohemocytes in larval lymph glands. Antibody staining revealed uniform H2AV expression across lymph gland zones, with phosphorylation (γ-H2AV) rarely observed under normal conditions .

  • Phenotype rescue: Expressing H2AV-RFP in mutants restores normal hemocyte differentiation, confirming H2AV’s role in prohemocyte maintenance .

DNA Damage Response

• γ-H2AV as a DSB marker:
  Monoclonal γ-H2AV antibodies detect DSBs in Drosophila oocytes and somatic cells. For example, irradiation induces γ-H2AV foci, which colocalize with repair proteins like Rad51 .

Table 2: Functional Domains of H2AV

• Chromatin remodeling: H2Av facilitates PARP1 binding to nucleosomes, promoting chromatin relaxation and transcription .

• Heterochromatin maintenance: H2Av depletion reduces H3K9 methylation and HP1 recruitment, leading to heterochromatin defects .

Technical Considerations

• Specificity: Rockland’s anti-H2AvD pS137 shows minimal cross-reactivity with non-phosphorylated H2AV .

• Optimal dilutions:

  • IF/IHC: 2–5 µg/mL (mouse antibodies) or 0.2–0.5 µg/mL (rabbit antibodies) .

  • WB: 0.2–0.5 µg/mL (mouse) or 20–50 ng/mL (rabbit) .