BCAT1 Antibody

What is BCAT1 and why is it significant in research applications?

BCAT1, also known as BCT1, is a cytosolic enzyme that catalyzes the first step in branched-chain amino acid (BCAA) catabolism. It's highly expressed during early embryogenesis and organogenesis, particularly in the neural tube, somites, and mesonephric tubules. Research significance stems from its expression in several MYC-based tumors and its chromosome 12 mapping . BCAT1 plays a critical regulatory role in cellular metabolism, particularly in immune cells like macrophages, where it forms the hub of a co-expression network in inflammatory diseases . Its dysregulation has been linked to cancer, metabolic disorders, and neurological conditions, making BCAT1 antibodies essential tools for investigating these pathways .

What experimental applications are BCAT1 antibodies validated for?

BCAT1 antibodies have been validated for multiple research applications:

• Western blotting (WB): Allows detection of BCAT1 protein expression levels in cell and tissue lysates, with expected molecular weight around 43 kDa .

• Immunohistochemistry (IHC): Enables visualization of BCAT1 distribution in tissue sections, as demonstrated in human ovarian adenoma and pancreatic cancer tissues .

• Metabolic studies: Useful for investigating BCAT1's role in BCAA metabolism and metabolic reprogramming in various cell types .

• T cell function research: Valuable for examining BCAT1's involvement in CD8+ T cell activation and exhaustion mechanisms .

How should researchers evaluate cross-reactivity when selecting BCAT1 antibodies?

Cross-reactivity evaluation is crucial for experimental validity. When selecting BCAT1 antibodies, researchers should:

• Check the immunogen sequence: Antibodies raised against human BCAT1 C-terminus might differ from mouse sequence by one amino acid and rat sequence by three amino acids, potentially affecting cross-reactivity .

• Examine validation data: Review manufacturer-provided Western blot and IHC images across different cell lines and tissues to assess specificity .

• Test in pilot experiments: For unstudied species (e.g., feline tissues), preliminary validation experiments are essential, as cross-reactivity is possible but not guaranteed without empirical testing .

• Consider blocking peptides: Using blocking peptides alongside primary antibodies can help confirm specificity, especially when working with closely related protein isoforms like BCAT1 and BCAT2 .

What are the optimal storage and handling conditions for BCAT1 antibodies?

For maximum stability and performance, BCAT1 antibodies require specific storage conditions:

When handling reconstituted antibodies:

• Reconstitute with distilled water to achieve 500 μg/ml concentration

• Mix gently to avoid protein denaturation

• For long-term storage, prepare small aliquots to minimize freeze-thaw cycles

• Always centrifuge briefly before opening the vial to collect all material

How should researchers optimize Western blot protocols for BCAT1 detection?

Based on validated protocols, the following optimization steps are recommended:

• Sample preparation:

  • Use 30 μg of total protein per lane

  • Prepare under reducing conditions

• Electrophoresis parameters:

  • Use 5-20% SDS-PAGE gradient gels

  • Run at 70V (stacking)/90V (resolving) for 2-3 hours

• Transfer conditions:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk/TBS for 1.5 hours at room temperature

• Antibody incubation:

  • Primary: Use BCAT1 antibody at 0.5 μg/mL overnight at 4°C

  • Washing: TBS-0.1% Tween, 3 times, 5 minutes each

  • Secondary: Goat anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hours at room temperature

• Detection:

  • Use enhanced chemiluminescence (ECL) detection systems

  • Expect a specific band at approximately 43 kDa

What are the critical parameters for immunohistochemical detection of BCAT1?

For optimal IHC results when studying BCAT1 expression in tissues:

• Antigen retrieval:

  • Heat-mediated retrieval in EDTA buffer (pH 8.0)

  • Complete retrieval is essential for accessing the BCAT1 epitope

• Blocking:

  • 10% goat serum is effective for reducing background

  • Duration and temperature affect background reduction

• Antibody concentrations:

  • Primary: 2 μg/ml rabbit anti-BCAT1 antibody

  • Incubation: Overnight at 4°C for optimal antigen binding

• Secondary detection:

  • Peroxidase-conjugated goat anti-rabbit IgG

  • Incubate for 30 minutes at 37°C

  • Develop with DAB chromogen for visualization

How can BCAT1 antibodies be used to investigate metabolic reprogramming in immune cells?

BCAT1 antibodies are valuable tools for studying metabolic changes in immune cells:

• Expression analysis in activated macrophages:

  • BCAT1 is the predominant BCAT isoform in human primary macrophages

  • Western blotting can track BCAT1 upregulation during macrophage activation

• Correlating BCAT1 with TCA cycle markers:

  • BCAT1 regulates HIF-1α, IL-1β, and IRG1 expression

  • Dual staining with BCAT1 antibodies and TCA cycle markers can reveal metabolic discontinuities at the Isocitrate Dehydrogenase 1 (IDH1) level

• Investigating leucine catabolism:

  • BCAT1 antibodies can help track how leucine metabolism through BCAT1 regulates IRG1-dependent macrophage activation

  • This pathway influences fatty acid production and itaconate synthesis

• Co-localization studies:

  • IHC with BCAT1 antibodies in inflammatory disease models can identify infiltrating macrophages where BCAT1 is highly expressed, such as in crescentic glomerulonephritis or rheumatoid arthritis tissues

What insights can BCAT1 antibodies provide in T cell function and cancer immunology research?

BCAT1 antibodies offer significant value in investigating T cell biology:

• T cell activation analysis:

  • Western blotting with BCAT1 antibodies can track expression changes during CD8+ T cell activation

  • Flow cytometry using BCAT1 antibodies can identify T cell populations with high BCAT1 expression

• Exhaustion phenotype studies:

  • Single-cell RNA-seq data link Bcat gene expression to exhausted T cells in tumor microenvironments

  • BCAT1 antibodies can help validate this connection at the protein level

• BCAT1 inhibition effects:

  • Combining BCAT1 antibody detection with inhibitor (e.g., ERG245) treatment allows researchers to correlate BCAT1 protein levels with functional outcomes

  • This approach has revealed that BCAT1 inhibition affects effector biomarkers like IFNγ and granzyme B in activated CD8+ T cells

• Iron homeostasis mechanism:

  • BCAT1 antibodies can help investigate the novel finding that BCAT1 functions through a mechanism controlling iron homeostasis without significantly altering BCAA levels

How do researchers differentiate between BCAT1 and BCAT2 during experimental analysis?

Distinguishing between these isoforms requires careful experimental design:

• Antibody selection criteria:

  • Choose antibodies raised against regions with minimal sequence homology

  • Verify isoform specificity through manufacturer validation data

• Expression pattern analysis:

  • BCAT1 (cytosolic) and BCAT2 (mitochondrial) have distinct subcellular localizations

  • Use subcellular fractionation followed by Western blotting with isoform-specific antibodies to confirm localization

• Comparative expression assessment:

  • RNA-sequencing of human monocyte-derived macrophages shows markedly higher BCAT1 mRNA levels compared to BCAT2

  • Protein-level verification with isoform-specific antibodies can validate this differential expression

• Inhibitor specificity:

  • ERG240 inhibits BCAT1 with an IC50 of 0.1-1 nM but shows no inhibition of BCAT2

  • Using such inhibitors alongside antibody detection can help differentiate isoform-specific functions

What are common issues encountered with BCAT1 antibodies and how can they be resolved?

Researchers frequently encounter these challenges:

• Weak signal intensity:

  • Increase antibody concentration incrementally (start with 2-fold increase)

  • Extend primary antibody incubation time (up to 48 hours at 4°C)

  • Optimize antigen retrieval for IHC (test different buffers: citrate pH 6.0 vs. EDTA pH 8.0)

  • Enhanced detection systems may be required for low-expressing samples

• Background or non-specific binding:

  • Increase blocking stringency (5% to 10% serum or BSA)

  • Reduce primary antibody concentration

  • Add 0.1-0.3% Triton X-100 for better penetration in IHC

  • Include additional washing steps with agitation

• Inconsistent results between experiments:

  • Standardize protein loading (verify with housekeeping controls)

  • Prepare fresh working solutions of antibodies

  • Document and maintain consistent incubation times and temperatures

  • Use the same batch of antibody when possible for comparative studies

• Species cross-reactivity issues:

  • For unstudied species, conduct preliminary validation experiments

  • Consider sequence homology when interpreting results across species

How should researchers interpret conflicting BCAT1 expression data in different experimental models?

When encountering contradictory results:

• Evaluate antibody specificity:

  • Confirm antibody recognizes the correct epitope using positive and negative controls

  • Test multiple BCAT1 antibodies targeting different epitopes to verify findings

• Consider methodological differences:

  • RNA vs. protein detection methods may yield different results

  • Post-translational modifications might affect antibody binding

  • Subcellular localization of BCAT1 can vary by cell type, affecting detection

• Biological context analysis:

  • BCAT1 expression varies significantly between tissues (e.g., high in ovarian adenoma and pancreatic cancer)

  • Cell activation state influences expression (e.g., LPS activation in macrophages)

  • Microenvironmental factors may alter expression patterns (e.g., tumor microenvironment for T cells)

• Integrated analysis approach:

  • Combine antibody-based detection with functional assays

  • Correlate with metabolic measurements

  • Validate with genetic approaches (siRNA, CRISPR) to confirm specificity

What considerations are important when studying BCAT1 in relation to therapeutic targeting?

Researchers exploring BCAT1 as a therapeutic target should consider:

• Inhibitor specificity verification:

  • Use BCAT1 antibodies to confirm target engagement after inhibitor treatment

  • Western blotting can detect potential compensatory upregulation of BCAT2 or related pathways

• Temporal dynamics:

  • ERG245 inhibition of BCAT1 in CD8+ T cells shows reversible effects

  • After withdrawal, cells can develop increased cytotoxicity, suggesting potential for temporal inhibition strategies in cancer immunotherapy

• Tissue-specific effects:

  • BCAT1 antibodies can help map expression in different disease contexts

  • Important for predicting on-target effects in specific tissues vs. potential off-target effects

• Biomarker development:

  • BCAT1 expression correlates with weight regain in human adipose tissue

  • BCAT1 antibodies could potentially aid in developing predictive biomarkers for treatment response