Acetyl-HIST1H2BB (K5) Antibody
Description
Applications
The antibody is validated for diverse experimental approaches:
Key Experimental Findings
-
ChIP Experiments: In HeLa cells treated with sodium butyrate (a histone deacetylase inhibitor), the antibody successfully immunoprecipitated chromatin associated with β-Globin promoter regions, confirming specificity for K5-acetylated HIST1H2BB .
-
Western Blot: Demonstrated increased acetylation in TSA-treated NIH/3T3 and C6 cells, with clear bands at ~14 kDa (HIST1H2BB molecular weight) .
-
Immunohistochemistry: Staining in rat spleen and human gastric cancer tissues showed nuclear localization, consistent with histone acetylation patterns .
Dilution Recommendations
Cross-Reactivity and Specificity
Research Implications
The Acetyl-HIST1H2BB (K5) Antibody is pivotal in studying:
-
Epigenetic Regulation: Links K5 acetylation to chromatin accessibility and transcriptional activation.
-
Cancer Biology: Investigates acetylation patterns in tumors, such as gastric cancer .
-
Drug Response: Assesses HDAC inhibitor efficacy (e.g., sodium butyrate, TSA) in modulating histone acetylation .
Critical Considerations
Product Specs
Components: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Histone H2B is a core component of the nucleosome, a fundamental unit of chromatin structure. Nucleosomes package and compact DNA, regulating access for cellular machinery involved in transcription, DNA repair, replication, and maintenance of chromosomal stability. This regulation is achieved through a complex interplay of post-translational histone modifications, often referred to as the histone code, and nucleosome remodeling.
- Studies have revealed functional crosstalk between histone H2B ubiquitylation and modifications/variants of H2A. PMID: 29643390
- Research indicates that RNF20 and H2Bub1 contribute to chronic colonic inflammation and inflammation-associated colorectal cancer in both mice and humans, partially through increased NF-κB activity and suppression of the antitumoral T cell response. PMID: 26854224
- RNF20-mediated H2B ubiquitination at DNA double-strand breaks (DSBs) plays a crucial role in homologous recombination repair (HRR) via chromatin remodeling. PMID: 21362548
Q&A
How should researchers validate Acetyl-HIST1H2BB (K5) antibody specificity across experimental applications?
Validation requires a multi-step approach combining peptide competition assays, isotype controls, and cross-application consistency checks. For ELISA, compare signals between acetylated and non-acetylated histone extracts, using peptide sequences matching the immunogen (residues surrounding acetyl-K5) to competitively inhibit antibody binding . In ICC/IF, validate nuclear localization patterns against negative controls (e.g., siRNA-mediated HIST1H2BB knockdown cells) . Cross-application validation is critical: a study using this antibody for ChIP in sodium butyrate-treated Hela cells confirmed specificity through >10-fold enrichment of β-globin promoter DNA compared to IgG controls .
Table 1: Validated Applications and Conditions
| Application | Dilution | Key Controls | Reference Study |
|---|---|---|---|
| ELISA | 1:1000 | Acetylated vs. non-acetylated lysates | |
| ICC/IF | 1:100 | siRNA knockdown + isotype-matched IgG | |
| ChIP | 8 µg/IP | Normal rabbit IgG + no-antibody chromatin |
What experimental controls are essential when quantifying histone acetylation via ICC/IF?
Three controls are mandatory:
-
Isotype control: Use rabbit IgG at the same concentration as the primary antibody to assess non-specific binding .
-
Epigenetic modulation control: Treat cells with histone deacetylase inhibitors (e.g., 30 mM sodium butyrate for 4 hours) to enhance acetylation signals, establishing assay dynamic range .
-
Competitive peptide blocking: Pre-incubate the antibody with 10x molar excess of immunogen peptide; signal reduction >80% confirms specificity .
How is the optimal antibody dilution determined for chromatin immunoprecipitation?
ChIP dilution depends on chromatin abundance and crosslinking efficiency. A tiered approach is recommended:
-
Start with 2–10 µg antibody per 1×10⁶ cells, as validated in Hela cells using 8 µg/IP .
-
Perform qPCR on immunoprecipitated DNA for a positive control locus (e.g., actively transcribed promoters). Signal-to-noise ratios >5:1 (antibody vs. IgG) indicate sufficient dilution . Adjust based on chromatin shearing efficiency: longer sonication fragments (>500 bp) may require higher antibody concentrations.
How can cross-reactivity with non-target acetylated histones be resolved in western blot assays?
Cross-reactivity often stems from antibody recognition of structurally similar acetyl-epitopes. Mitigation strategies include:
-
2D gel electrophoresis: Resolve histone variants by isoelectric point before blotting; Acetyl-HIST1H2BB (K5) should migrate at pI 10.2–10.5 .
-
Mutant cell lines: Use CRISPR-edited cells lacking HIST1H2BB but retaining other H2B variants. Loss of signal confirms specificity .
-
Liquid chromatography tandem mass spectrometry (LC-MS/MS): Directly identify immunoprecipitated proteins to detect off-target binding .
What protocol adjustments optimize ChIP for low-abundance acetylated targets?
Key modifications derived from lymphoma cell studies include:
-
Dual crosslinking: Initial formaldehyde fixation (1% for 10 min) followed by disuccinimidyl glutarate (DSG; 2 mM for 45 min) improves histone-antibody complex stability .
-
Micrococcal nuclease (MNase) titration: Digest chromatin to mononucleosome-sized fragments (150–200 bp) using 4–8 U MNase/µg DNA, verified by agarose gel electrophoresis .
-
Signal amplification: Combine tyramide-based amplification with 1:500 secondary antibody dilution, increasing sensitivity 10-fold for rare acetylated loci .
Table 2: Optimized ChIP Conditions for Low-Abundance Targets
| Parameter | Standard Protocol | Optimized Protocol |
|---|---|---|
| Crosslinking | 1% formaldehyde | Formaldehyde + DSG |
| Chromatin digestion | 2 U MNase/µg DNA | 8 U MNase/µg DNA |
| Antibody incubation | 4°C overnight | 24 hr with rotation |
How should researchers address contradictory acetylation levels observed across cell types?
Contradictions often arise from cell-cycle-dependent acetylation or technical artifacts. A systematic framework is recommended:
-
Synchronize cell cycles: Compare G1-arrested (serum starvation) vs. S-phase (double thymidine block) cells using flow cytometry. Acetyl-HIST1H2BB (K5) levels typically peak in S-phase .
-
Quantify histone turnover: Pulse-chase experiments with labeled amino acids (e.g., ¹³C-lysine) distinguish synthesis-dependent acetylation changes .
-
Normalize to total H2B: Measure HIST1H2BB mRNA (RT-qPCR) and protein (pan-H2B ELISA) to rule out expression-level confounders .
Methodological Considerations for Data Interpretation
-
Batch variability: Aliquot antibodies to minimize freeze-thaw cycles; signal drops >20% after 5 thaws indicate reagent degradation .
-
Epigenetic crosstalk: Co-stain for H3K27me3 or H4K16ac to identify confounding modifications; spatial proximity (≤50 nm) confirmed by proximity ligation assay (PLA) may artificially elevate signals .
-
Quantitative thresholds: In ChIP-qPCR, define positivity as ≥3 standard deviations above IgG control. For heterogeneous samples (e.g., tumor biopsies), single-cell ATAC-seq can stratify acetylation heterogeneity .
