AHCY Antibody

What are the validated applications for AHCY antibodies in molecular research?

AHCY antibodies have been extensively validated for multiple experimental applications with specific dilution recommendations:

For optimal results, perform antibody titration in each specific testing system. AHCY antibodies have demonstrated successful reactivity across human, mouse, rat, pig, and branchiostoma belcheri samples . When using previously untested sample types, validation experiments are recommended to confirm specificity.

What is the optimal protocol for Western blot detection of AHCY?

For effective Western blot detection of AHCY:

• Prepare cell/tissue lysates (validated sources: COLO 320, HepG2, HeLa, Jurkat cells, mouse/rat liver tissue)

• Load samples on 12% SDS-PAGE gels

• Run electrophoresis (90 minutes at 120V is commonly used)

• Transfer proteins to PVDF membrane

• Block with 5% nonfat milk in TBST

• Incubate overnight with primary AHCY antibody (1:2000-1:10000 dilution)

• Wash and apply appropriate secondary antibody (e.g., goat anti-rabbit HRP at 1:5000)

• Look for specific band at 45-48 kDa

For quantitative Western blot analysis, include loading controls or standardize based on co-transfected markers such as β-galactosidase to correct for transfection efficiency variations . AHCY antibodies recognize the protein across multiple species, enabling comparative studies between human and common model organisms .

How should researchers control for AHCY antibody specificity in knockdown/knockout experiments?

Proper validation of AHCY antibody specificity in knockdown/knockout experiments requires:

• CRISPR-Cas9 knockout validation: Use lentiCRISPRv2 vector with guide RNA targeting AHCY exon 4 (5′-tgcgcacctgacagaagctg-3′) and include EGFP-targeting guide RNA as control .

• shRNA knockdown design: Develop inducible shRNA systems using doxycycline for controlled expression to verify antibody specificity through gradual protein reduction .

• Genetic rescue experiments: To confirm specificity, reintroduce WT or mutant (e.g., K186N) AHCY in knockdown cells and assess antibody signal recovery .

• Immunoblotting controls: AHCY ChIP signals should diminish at specific promoters following knockdown, providing functional validation of antibody specificity .

• Genomic verification: Confirm knockout by Sanger sequencing of PCR amplicons using primers:

  • Forward: 5′-CACCTCCTCACCAATGTCCT-3′

  • Reverse: 5′-CTGTGGTGCATTGAGCAGAC-3′

These approaches provide multi-level confirmation that observed signals are specifically due to AHCY protein binding rather than non-specific interactions.

What controls should be included when using AHCY antibodies for immunohistochemistry?

When conducting immunohistochemistry with AHCY antibodies, implement these essential controls:

• Negative controls:

  • Omit primary antibody (secondary only)

  • Use isotype-matched control antibodies (rabbit IgG)

  • Include tissue known to lack AHCY expression

  • Use AHCY knockdown/knockout tissue sections when available

• Positive controls:

  • Include validated human colon cancer tissue samples

  • Use tissues with known high AHCY expression (liver, kidney, thyroid)

• Antigen retrieval optimization:

  • Compare TE buffer pH 9.0 (recommended) with citrate buffer pH 6.0

  • Titrate antibody dilutions between 1:500-1:2000 for each tissue type

• Cross-reactivity assessments:

  • Validate species specificity when working with non-human samples

  • Perform peptide competition assays with immunogen peptide

• Developmental stage considerations:

  • For embryonic studies, AHCY shows nuclear localization in NANOG-positive cells at the blastula stage

Proper controls ensure accurate interpretation of AHCY localization patterns, particularly important when examining tissues with varying expression levels.

How can AHCY antibodies be used to investigate chromatin association and epigenetic regulation?

AHCY antibodies provide powerful tools for investigating chromatin association and epigenetic regulation:

• ChIP-seq methodology:

  • Cross-link protein-DNA complexes with formaldehyde

  • Sonicate chromatin to 200-500bp fragments

  • Immunoprecipitate with AHCY antibody

  • Process DNA for next-generation sequencing

  • Analyze using MACS software with adjusted shift size of 100bp

  • Select peaks with fold-enrichment scores above 4.50 as confident binding sites

• AHCY chromatin localization patterns:

  • AHCY preferentially localizes to transcription start sites (TSS)

  • Target genes typically display transcriptionally permissive histone modifications (H3K36me3, H3K4me3, H3K27ac)

  • AHCY occupancy correlates with cancer-related ESC-specific Myc network factors

• Functional genome analysis:

  • AHCY antibodies reveal enrichment at highly expressed genes in ESCs

  • Target genes frequently include ribosomal protein genes

  • AHCY recruitment to chromatin increases during replication and transcription

These approaches help elucidate AHCY's role beyond its enzymatic function, revealing its direct participation in gene regulation through chromatin association.

How do AHCY antibodies contribute to understanding the link between metabolism and gene regulation?

AHCY antibodies reveal crucial connections between cellular metabolism and gene regulation:

• Circadian rhythm connections:

  • AHCY associates with CLOCK-BMAL1 at chromatin, linking methionine metabolism to circadian regulation

  • Use immunoprecipitation with AHCY antibodies followed by proteomics to identify temporal interaction partners

• Methylation pathway analysis:

  • AHCY inhibition with 3-deazaadenosine (3-DZA) affects protein synthesis

  • Study effects through L-homopropargylglycine (L-HPG) incorporation assays

  • Combine with AHCY immunoblotting to correlate enzyme levels with methylation activity

• Stem cell pluripotency regulation:

  • AHCY expression increases with acquisition of pluripotency

  • Use AHCY antibodies in immunostaining to visualize nuclear localization in NANOG-positive cells

  • Apply AHCY ChIP-seq to map binding sites at ribosomal protein genes

• Molecular interaction studies:

  • Use AHCY antibodies to investigate NAD+/NADH ratio effects on protein function

  • Examine post-translational modifications like acetylation at lysines 401 and 408

These approaches illuminate how AHCY serves as a molecular bridge between cellular metabolism and gene expression regulation, particularly in pluripotent cells and during development.

What are common pitfalls when using AHCY antibodies and how can they be addressed?

Researchers frequently encounter these challenges when working with AHCY antibodies:

• Cross-reactivity issues:

  • Problem: False positive signals from related proteins

  • Solution: Validate with knockout/knockdown controls; use antigen-affinity purified antibodies

• Epitope masking:

  • Problem: Post-translational modifications may block antibody binding

  • Solution: Compare antibodies targeting different AHCY epitopes; use multiple antibodies targeting different regions

• Isoform specificity:

  • Problem: AHCY has three annotated splicing isoforms (432, 404, and 434 amino acids)

  • Solution: Verify which isoforms your antibody detects; select antibodies that distinguish between N-terminal variants

• Tetrameric structure interference:

  • Problem: AHCY forms homotetramers that may obscure epitopes

  • Solution: Ensure proper sample denaturation for Western blot; consider native conditions for IP

• Nuclear vs. cytoplasmic localization:

  • Problem: AHCY distributes differentially between compartments

  • Solution: Use subcellular fractionation; compare with known nuclear and cytoplasmic markers

Addressing these challenges requires careful antibody selection and experimental design, particularly when studying AHCY's diverse cellular functions.

How can researchers optimize AHCY antibody storage and handling for maximum sensitivity?

To maintain AHCY antibody performance and sensitivity:

For optimal experimental performance:

• Determine application-specific optimal dilutions through titration

• Store working dilutions at 4°C for short term use only

• For immunohistochemistry, verify antigen retrieval conditions (TE buffer pH 9.0 or citrate buffer pH 6.0)

• When using for the first time with a new sample type, include positive and negative controls

• For quantitative applications, prepare fresh dilutions for each experiment

Following these guidelines ensures consistent antibody performance across experiments and maximizes detection sensitivity.

How can AHCY antibodies be used to investigate the relationship between AHCY and disease models?

AHCY antibodies offer valuable insights into disease mechanisms:

• AHCY deficiency studies:

  • Use antibodies to confirm reduced AHCY levels in hypermethioninemia models

  • Compare tissue expression patterns with developmental and neurological phenotypes

  • Perform immunohistochemistry on liver, muscle, and CNS tissues to track pathological progression

• Cancer research applications:

  • Analyze AHCY expression in cancer cell lines (COLO 320, HepG2, HeLa, Jurkat)

  • Compare with matched normal tissues to identify expression changes

  • Correlate with Myc network activity through co-immunoprecipitation

• Metabolic disorder investigations:

  • Track AHCY levels during methionine metabolism perturbations

  • Combine with S-adenosylmethionine/S-adenosylhomocysteine ratio measurements

  • Monitor methyltransferase inhibition consequences

• Developmental disorder models:

  • Use antibodies to detect AHCY during embryogenesis

  • Correlate with NANOG expression in pluripotent cells

  • Track protein synthesis rates using L-HPG incorporation assays in control vs. treated embryos

AHCY antibodies facilitate mechanistic studies connecting this metabolic enzyme to broader disease processes, highlighting potential therapeutic targets.

What approaches can be used to study AHCY post-translational modifications using antibodies?

Post-translational modifications (PTMs) of AHCY can be studied through:

• Acetylation analysis:

  • Mammalian AHCY is acetylated at lysines 401 and 408

  • Use acetylation-specific antibodies alongside total AHCY antibodies

  • Compare acetylation status across different metabolic conditions

• NAD+/NADH ratio effects:

  • AHCY activity is influenced by NAD+/NADH binding

  • Combine with activity assays to correlate PTMs with functional changes

  • Investigate adenosine binding site usage under different cofactor conditions

• Phosphorylation mapping:

  • Use phospho-specific antibodies in conjunction with total AHCY antibodies

  • Apply phosphatase treatments as controls

  • Identify cell cycle-dependent modifications

• Immunoprecipitation-mass spectrometry approach:

  • Immunoprecipitate AHCY using validated antibodies

  • Analyze by mass spectrometry to identify novel PTMs

  • Confirm findings with site-specific antibodies or mutagenesis studies

These approaches help decipher how AHCY function is regulated post-translationally, potentially revealing additional layers of control beyond transcriptional regulation.

How are AHCY antibodies being used to explore chromatin regulation in stem cell biology?

Recent studies reveal AHCY's unexpected role in stem cell regulation through chromatin:

• ESC proliferation mechanisms:

  • AHCY knockdown reduces S phase cells and increases G1 phase

  • Antibodies reveal AHCY recruitment to ribosomal protein genes

  • ChIP-seq identifies AHCY binding preferences near TSS regions of highly expressed genes

• Early developmental regulation:

  • AHCY expression increases with blastomere pluripotency acquisition

  • Immunostaining shows nuclear localization in NANOG-positive cells

  • Antibodies help track AHCY during preimplantation development

• Protein synthesis connection:

  • AHCY depletion reduces ribosomal proteins and protein synthesis

  • Combined proteomic and immunoblotting analyses confirm this link

  • AHCY inhibition with 3-DZA causes developmental growth delay

• Myc network association:

  • AHCY occupancy correlates with cancer-related ESC-specific Myc network

  • Antibodies help identify co-regulatory factors (TRIM28, DMAP1)

  • ChIP-seq reveals correlation with permissive histone modifications

These findings establish AHCY as a critical regulator of pluripotent cell proliferation through direct chromatin association, revealing functions beyond its classical metabolic role.

What are advanced techniques for studying AHCY enzyme-protein interactions using antibodies?

To investigate AHCY's complex protein interaction network:

• Proximity ligation assays (PLA):

  • Visualize AHCY interactions with suspected partners in situ

  • Combine with cell synchronization to detect cell cycle-dependent interactions

  • Use with CLOCK-BMAL1 to confirm circadian rhythm connections

• ChIP-reChIP approaches:

  • Perform sequential ChIP with AHCY antibody followed by partner antibodies

  • Identify genomic regions with co-occupancy of AHCY and transcription factors

  • Map co-regulatory complexes at specific target genes

• BioID or APEX2 proximity labeling:

  • Generate AHCY fusion constructs with biotin ligase

  • Use antibodies to validate interaction candidates

  • Map protein interaction networks in different cellular compartments

• FRET/FLIM microscopy:

  • Label AHCY and potential partners with appropriate fluorophores

  • Use antibodies for immunofluorescence validation

  • Measure real-time interactions in living cells

These sophisticated techniques move beyond simple co-immunoprecipitation to study dynamic AHCY interactions in native cellular contexts, providing insights into its multifaceted roles in metabolism and gene regulation.

How can AHCY antibodies contribute to understanding metabolic pathways in disease?

AHCY antibodies provide valuable tools for metabolic disease research:

• Transmethylation pathway analysis:

  • AHCY deficiency impairs the conversion of S-adenosylhomocysteine to homocysteine and adenosine

  • Use antibodies to track enzyme levels in patient samples

  • Correlate with methionine/homocysteine levels and methylation patterns

• One-carbon metabolism disruptions:

  • AHCY is a competitive inhibitor of S-adenosyl-L-methionine-dependent methyl transferases

  • Antibodies help monitor enzyme levels during metabolic perturbations

  • Use in combination with methyltransferase activity assays

• Therapeutic intervention assessment:

  • Track AHCY levels during experimental treatments

  • Monitor subcellular localization changes following intervention

  • Correlate with clinical parameters in model systems

• Multi-tissue expression profiling:

  • AHCY is expressed ubiquitously but with higher levels in kidney and thyroid

  • Use immunohistochemistry to map expression in disease states

  • Compare with normal tissue expression patterns

These approaches help unravel the complex relationships between AHCY dysfunction and various metabolic disorders, potentially leading to new therapeutic strategies.

What considerations are important when using AHCY antibodies in genetic variant studies?

When investigating AHCY genetic variants:

• Allozyme detection strategies:

  • Human AHCY gene contains multiple polymorphisms

  • Use quantitative Western blot analysis to compare variant protein levels

  • Control for transfection efficiency with co-transfected markers (β-galactosidase)

• Promoter variant analysis:

  • Approximately 500 bp of the AHCY 5′-FR contains functional polymorphisms

  • Use reporter assays in conjunction with antibodies to correlate variant expression

  • Compare results across cell types (HEK293T, HepG2)

• Functional impact assessment:

  • Compare antibody detection of wild-type versus variant AHCY proteins

  • Assess subcellular localization differences

  • Correlate with enzymatic activity measurements

• Ethnicity considerations:

  • AHCY variants differ across ethnic groups

  • Consider population-specific polymorphisms in study design

  • Use appropriate controls matched for genetic background