BACH1 Antibody

What is BACH1 and why is it important in research?

BACH1 is a master transcriptional repressor in heme homeostasis and antioxidant defense systems. As a member of the basic leucine zipper (bZIP) protein family, BACH1 heterodimerizes with small musculoaponeurotic fibrosarcoma (sMAF) proteins and binds to antioxidant response elements (AREs) of many antioxidant genes, suppressing their transcription under unstressed conditions . BACH1 has been implicated in various disease processes including cancer, sickle cell disease, and inflammatory conditions.

The protein is structurally characterized by:

• BTB (Broad-Complex, Tramtrack and Bric-à-brac) domain for protein dimerization

• CNC (cap 'n' collar) domain for DNA binding

• Multiple cysteine residues that serve as heme-binding sites

• Reported molecular weight of 82 kDa, though often observed at 100-110 kDa in Western blots

How do I select the appropriate BACH1 antibody for my research application?

Selection criteria should focus on:

• Target specificity: Ensure the antibody specifically recognizes BACH1 and not its paralog BACH2. Some antibodies may cross-react with BACH2, which shares 62% sequence identity in the BTB domain .

• Validated applications: Match the antibody to your intended application. From the search results, BACH1 antibodies are available for:

  • Western blotting (WB)

  • Immunohistochemistry (IHC)

  • Immunofluorescence (IF)

  • Chromatin immunoprecipitation (ChIP)

  • Immunoprecipitation (IP)

  • Flow cytometry

• Species reactivity: Verify reactivity with your experimental model. Most commercial antibodies react with human BACH1, while some also recognize mouse and rat orthologs .

• Epitope location: Consider antibodies targeting different regions of BACH1, as epitope accessibility may vary depending on BACH1's interactions with other proteins or post-translational modifications.

How do I avoid confusion between BACH1 (transcription factor) and BACH1/BRIP1 (DNA helicase)?

A critical issue in BACH1 research is the existence of two distinct proteins both referred to as "BACH1" in the literature:

• BACH1 (BTB and CNC homology 1): The transcription factor involved in heme homeostasis and antioxidant response (Gene ID: 571) .

• BACH1/BRIP1/FANCJ: A DNA helicase that interacts with BRCA1 and is involved in DNA repair (BRCA1-interacting protein C-terminal helicase 1) .

To avoid confusion:

• Verify which BACH1 protein your antibody targets by checking the gene ID or UniProt accession number (O14867 for transcription factor BACH1)

• Be explicit in publications about which BACH1 protein you're studying

• Consider using alternative names (BRIP1 or FANCJ) for the DNA helicase to avoid ambiguity

• Check molecular weight (transcription factor BACH1 is typically observed at 100-110 kDa, while BRIP1/FANCJ is approximately 130 kDa)

What are the optimal conditions for Western blot detection of BACH1?

Based on the compiled research data:

• Sample preparation:

  • Cell lines with verified BACH1 expression: HEK-293, MCF-7, Raji, HeLa, Jurkat, K-562

  • Mouse cell lines: NIH-3T3

  • Use reducing conditions for consistent results

• Antibody dilutions:

  • Primary antibody: 1:5000-1:50000 (polyclonal)

  • Monoclonal antibodies: typically 1:1000-1:2000

• Detection considerations:

  • Expected molecular weight: Though the calculated molecular weight is 82 kDa, BACH1 typically appears at 100-110 kDa in Western blots

  • Use appropriate positive controls from validated cell lines

  • Consider using specific buffers: Immunoblot Buffer Group 1 has been validated for BACH1 detection

What methodology should I follow for BACH1 detection in tissue sections?

For immunohistochemical detection of BACH1:

• Tissue processing:

  • Fixed, paraffin-embedded sections perform well with BACH1 antibodies

  • Heat-induced epitope retrieval is critical for optimal staining

• Recommended protocol:

  • Antigen retrieval: Use TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)

  • Primary antibody concentration: 1:750-1:3000 dilution for polyclonal antibodies

  • For monoclonal antibodies, approximately 10 μg/mL has been validated

  • Counterstain with hematoxylin for nuclear contrast

• Validated tissue types:

  • Human lung cancer tissue

  • Human cervical cancer tissue

  • Human breast cancer and hyperplasia tissue

• Expected results:

  • BACH1 typically shows nuclear localization with some cytoplasmic expression

  • Expression patterns may vary by tissue type and disease state

How can I optimize chromatin immunoprecipitation (ChIP) assays using BACH1 antibodies?

ChIP assays are critical for studying BACH1's function as a transcriptional regulator:

• Target selection:

  • Known BACH1 binding sites include MAF recognition elements (MAREs) with the sequence 5′-TGACTCGCA-3′

  • Validated BACH1 target genes include HMOX1, FTH1, and mitochondrial genes like ATP5D, COX15, UQCRC1

• Cross-linking conditions:

  • Standard 1% formaldehyde for 10 minutes at room temperature

  • Consider dual cross-linking with disuccinimidyl glutarate (DSG) followed by formaldehyde for studying BACH1 complexes

• Antibody considerations:

  • Use ChIP-validated BACH1 antibodies

  • Recommended amount: 2-5 μg per immunoprecipitation

  • Include appropriate IgG controls

• Data analysis:

  • Compare BACH1 binding under different conditions (e.g., with/without heme, oxidative stress)

  • Remember that only about 11% of BACH1-binding sites overlap with MAFK-binding sites genome-wide

How do I design experiments to study heme-mediated regulation of BACH1?

BACH1 is regulated by heme levels, which affect its stability, localization, and DNA binding:

• Experimental approaches:

  • Treat cells with hemin (10-50 μM) to induce BACH1 degradation

  • Monitor BACH1 protein levels by Western blot

  • Track BACH1 nuclear export using immunofluorescence

  • Assess release from target gene promoters via ChIP

• Heme-resistant BACH1 mutants:

  • Generate cysteine-to-alanine mutations at C-terminal heme-binding sites

  • These mutants remain stable and active despite hemin treatment

  • Use as controls to confirm heme-specific effects

• Readouts for BACH1 activity:

  • Measure expression of BACH1 target genes (e.g., HMOX1, FTH1)

  • Monitor cellular metabolic parameters

  • Assess sensitivity to metabolic inhibitors like metformin

What are the methodological considerations for studying BACH1 in oxidative stress responses?

BACH1 is a critical regulator of antioxidant responses through its competition with NRF2:

• Experimental design:

  • Induce oxidative stress with H₂O₂, paraquat, or glutathione depletion

  • Use BACH1 inhibitors like ASP8731 alongside oxidative stressors

  • Compare responses in wild-type vs. BACH1-knockdown or knockout models

• Key measurements:

  • Glutathione levels: BACH1 inhibition protects against hemin-induced glutathione depletion

  • ROS production: BACH1 overexpression increases mitochondrial ROS

  • Expression of antioxidant genes: HMOX1, FTH1, NQO1

• Cell models:

  • Endothelial cells: Human pulmonary arterial endothelial cells (PAEC) show BACH1-dependent responses to TNF-α and hemin

  • Hepatocytes: HepG2 cells show robust BACH1-dependent antioxidant gene regulation

How can I investigate the therapeutic potential of BACH1 inhibition?

BACH1 inhibition shows promise in treating conditions like sickle cell disease (SCD) and potentially cancer:

• BACH1 inhibitor studies:

  • ASP8731 is a selective small molecule BACH1 inhibitor with characterized effects

  • Treatment protocol: Daily administration for 2-4 weeks (3-25 mg/kg in mouse models)

• Disease models:

  • For SCD: Townes-SS mice treated with BACH1 inhibitors show reduced microvascular stasis

  • For cancer studies: TNBC xenograft models show sensitivity to combined BACH1 degradation (via hemin) and metformin treatment

• Key endpoints to measure:

  • Gamma-globin expression and F-cell production in SCD models

  • Inflammatory markers: VCAM1, ICAM-1, NF-kB phospho-p65

  • Mitochondrial function: Oxygen consumption rate (OCR)

  • Tumor growth in cancer models

What techniques can be used to study BACH1 protein interactions and degradation?

Understanding BACH1 protein interactions is essential for deciphering its regulatory mechanisms:

• Protein-protein interaction methods:

  • Co-immunoprecipitation with BACH1 antibodies to identify interaction partners

  • Bioluminescence resonance energy transfer (BRET) or FRET for real-time interaction dynamics

  • Proximity ligation assay for visualizing interactions in situ

• Studying BACH1 degradation:

  • Cycloheximide chase experiments to measure BACH1 half-life

  • Proteasome inhibitors (MG132) to confirm proteasome-dependent degradation

  • Ubiquitination assays to detect poly-ubiquitinated BACH1

• BACH1 quaternary structure analysis:

  • Size-exclusion chromatography with multi-angle light scattering (SEC-MALS) to analyze BACH1 BTB domain dimerization

  • Bio-layer interferometry (BLI) to study interaction kinetics

  • Investigation of FBXO22 and FBXL17-mediated degradation pathways

How do I troubleshoot variability in BACH1 detection by Western blot?

BACH1 detection can be challenging due to several factors:

• Molecular weight variability:

  • Expected range: 100-110 kDa, despite calculated MW of 82 kDa

  • Post-translational modifications affect migration pattern

  • Use positive control lysates from validated cell lines (HEK-293, NIH-3T3)

• Protein stability issues:

  • BACH1 is rapidly degraded under certain conditions (e.g., high heme)

  • Include protease inhibitors in lysis buffers

  • Consider proteasome inhibitor treatment before cell collection

• Antibody specificity:

  • Verify antibody specificity using BACH1 knockdown or knockout controls

  • Test multiple antibodies targeting different epitopes

  • Be aware of potential cross-reactivity with BACH2

How should I interpret changes in BACH1 localization during experimental manipulations?

BACH1 subcellular localization provides important functional information:

• Normal localization pattern:

  • Predominantly nuclear under basal conditions

  • Some cytoplasmic expression may be observed

• Expected changes:

  • Increased cytoplasmic localization after heme treatment (indicates nuclear export)

  • Changes in nuclear/cytoplasmic ratio with oxidative stress

  • Altered localization with specific mutations (e.g., heme-binding site mutations)

• Technical considerations:

  • Use nuclear/cytoplasmic fractionation followed by Western blot for quantitative assessment

  • For immunofluorescence, include nuclear counterstain and z-stack imaging

  • Quantify nuclear/cytoplasmic signal ratios across multiple cells

How can I differentiate between effects on BACH1 expression versus activity?

BACH1 function can be regulated at multiple levels:

• Expression vs. activity markers:

  • Expression: Total BACH1 protein/mRNA levels

  • Activity: Target gene expression (e.g., HMOX1, FTH1)

  • DNA binding: ChIP assays at known target genes

• Post-translational regulation:

  • Heme binding induces conformational changes and nuclear export

  • Oxidative stress can modify BACH1 cysteine residues (particularly Cys107 and Cys122)

  • These modifications may not change total BACH1 levels but alter activity

• Experimental approach:

  • Compare changes in BACH1 protein levels with changes in target gene expression

  • Use reporter assays with BACH1-responsive promoters

  • Employ ChIP to directly measure BACH1 occupancy at target genes

What are the methodological approaches to study BACH1 in metabolic reprogramming?

Recent research has implicated BACH1 in cancer metabolic reprogramming:

• Experimental techniques:

  • Seahorse analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)

  • Metabolomics profiling to assess TCA cycle intermediates

  • 13C-glucose or 13C-glutamine tracing to track metabolic flux

  • Analysis of mitochondrial gene expression and function

• Combination treatment strategies:

  • BACH1 degradation via hemin sensitizes triple-negative breast cancer to metformin treatment

  • Monitor changes in metabolic flexibility when BACH1 is inhibited

  • Assess mitochondrial function in wild-type versus BACH1-null or inhibited cells

• Key findings to build upon:

  • BACH1 represses mitochondrial genes like ATP5D, COX15, UQCRC1

  • BACH1 inhibition reduces metabolic plasticity and increases dependence on mitochondrial respiration

  • Heme-resistant BACH1 mutants can serve as controls to confirm BACH1-specific effects

How can researchers investigate BACH1's role in immune cell differentiation?

BACH1 plays important roles in immune cell development and function:

• Experimental models:

  • BACH1 knockout mice show altered antigen-presenting cell development

  • Study differentiation of monocytes into red pulp macrophages with/without BACH1

  • Analyze T and B cell lineage development in BACH1-deficient models

• Key methodologies:

  • Flow cytometry to track immune cell populations and differentiation markers

  • Single-cell RNA sequencing to identify BACH1-dependent transcriptional programs

  • ChIP-seq to map BACH1 binding sites in different immune cell types

• Functional assays:

  • T-cell activation assays with BACH1-deficient antigen-presenting cells

  • Assessment of macrophage polarization (M1/M2) with BACH1 manipulation

  • In vivo immune challenge models (e.g., experimental autoimmune encephalomyelitis)

What strategies can be employed to study BACH1 and BACH2 functional differences?

BACH1 and BACH2 share structural similarities but have distinct functions:

• Comparative analysis approaches:

  • Generate isoform-specific knockdown/knockout models

  • Create chimeric proteins to map domain-specific functions

  • Compare tissue expression patterns (BACH1 is widely expressed; BACH2 primarily in brain and spleen)

• Distinguishing biochemical properties:

  • Perform ChIP-seq with isoform-specific antibodies to identify unique binding sites

  • Compare protein interaction partners through mass spectrometry

  • Assess differential responses to heme, oxidative stress, and other stimuli

• Technical considerations:

  • Validate antibody specificity against both isoforms

  • Use rescue experiments with isoform-specific expression constructs

  • Consider compensatory mechanisms in knockout models