BHMT Antibody

What is BHMT and why are antibodies against it important for research?

BHMT (betaine-homocysteine S-methyltransferase) is a 45 kDa protein consisting of 406 amino acid residues in humans with subcellular localization in both the nucleus and cytoplasm. It plays a critical role in regulating homocysteine metabolism by converting betaine and homocysteine to dimethylglycine and methionine, respectively .

BHMT antibodies are important research tools because:

• They enable detection and quantification of BHMT expression in various tissues

• They help researchers study the role of BHMT in one-carbon metabolism pathways

• They facilitate investigation of homocysteine-related disorders

• They allow examination of BHMT's role in choline metabolism, as the BHMT reaction is also required for irreversible oxidation of choline

BHMT is notably expressed in kidney and liver tissues, making antibodies particularly valuable for research in these organ systems .

What are the main applications of BHMT antibodies in research?

BHMT antibodies can be utilized in multiple experimental applications:

The optimal working dilution should be determined by each researcher based on their specific experimental conditions and antibody characteristics . For example, immunohistochemical analysis of paraffin-embedded human kidney tissue has been performed using anti-BHMT antibodies at dilutions as high as 1/20,000, followed by secondary antibody detection .

What is the difference between BHMT and BHMT2?

BHMT and BHMT2 are closely related proteins with distinct properties:

Some antibodies are designed to detect both BHMT and BHMT2, such as the recombinant monoclonal antibody [EPR20822] that recognizes both proteins .

How do genetic variants of BHMT affect antibody recognition and experimental outcomes?

Genetic variations in BHMT can potentially impact antibody recognition depending on where the variations occur and which epitopes the antibody targets. Research has identified multiple polymorphisms in the BHMT gene that have functional implications:

Functional genomic studies have been performed on BHMT variant allozymes and common 5′-flanking region haplotypes. These studies have shown that different BHMT variants may have altered stability, activity, or expression levels. When conducting experiments with BHMT antibodies, researchers should consider:

• Whether polymorphisms occur within the antibody's epitope region

• If variants affect protein folding, potentially masking the epitope

• Whether post-translational modifications associated with variants alter antibody binding

• The possibility of differential expression levels of variants affecting signal intensity

To address these concerns, researchers should:

• Validate antibody recognition across known BHMT variants relevant to their research

• Include appropriate controls when studying populations with polymorphic BHMT

• Consider using multiple antibodies targeting different epitopes for confirmation

• Document the specific BHMT genetic background in experimental models

What factors influence BHMT protein stability and how does this impact antibody-based detection methods?

BHMT protein stability can be influenced by multiple factors that researchers should consider when designing antibody-based detection experiments:

• Homocysteine levels: Research has shown that homocysteine can "stabilize" BHMT proteins. This has implications for sample preparation and experimental conditions—maintaining physiological homocysteine levels may preserve BHMT integrity .

• Protein-protein interactions: BHMT2 stability appears to be enhanced through interaction with BHMT. This interaction has been demonstrated through co-immunoprecipitation experiments where BHMT and HA-tagged BHMT2 coprecipitated during immunoprecipitation with anti-HA-agarose .

• Sample preparation conditions: The integrity of BHMT during cell lysis and processing can affect detection. For optimal results in Western blotting and immunoprecipitation, researchers should:

  • Use appropriate protease inhibitors

  • Control temperature during processing

  • Consider native vs. denaturing conditions based on experimental goals

  • Optimize buffer composition to maintain protein stability

• Antibody selection: Different antibodies may perform differently depending on the native or denatured state of BHMT. Validation of antibody performance under specific experimental conditions is essential .

How can BHMT antibodies be used to investigate the relationship between BHMT function and disease pathways?

BHMT antibodies serve as valuable tools for investigating connections between BHMT function and disease pathways through several methodological approaches:

• Expression analysis in disease models:

  • Comparing BHMT expression levels in normal versus diseased tissues using immunohistochemistry or Western blotting

  • Quantifying changes in BHMT expression during disease progression

  • Correlating BHMT expression with disease biomarkers

• Manipulation of BHMT expression:

  • Evaluating the consequences of BHMT knockdown using siRNA approaches, as demonstrated in mouse primary hepatocytes

  • The siRNA sequence 5′-GUGAAGACAAGCUGGAAAAd(TT)-3′ targeting BHMT has been successfully used in liver cells

  • Monitoring physiological and biochemical changes following BHMT modulation

• Investigation of protein interactions:

  • Using co-immunoprecipitation with BHMT antibodies to identify novel interaction partners

  • Analyzing how these interactions change in disease conditions

  • Immunoprecipitation protocols using anti-HA immunoprecipitation kits have been established for BHMT protein interaction studies

• Subcellular localization studies:

  • Examining changes in BHMT localization under different physiological or pathological conditions

  • Immunofluorescence analysis of BHMT in cell lines such as Hep3B has been established using dilutions around 1:100, combined with nuclear staining (DAPI)

What are the optimal protocols for using BHMT antibodies in Western blotting applications?

For optimal Western blotting using BHMT antibodies, researchers should consider the following methodological details:

Sample Preparation:

• Tissue samples: BHMT is highly expressed in liver and kidney tissues, making these ideal positive controls

• Cell lysates: Hepatic cell lines (HepG2, Hep3B) express detectable BHMT levels

• Protein extraction: Use RIPA buffer with protease inhibitors to preserve BHMT integrity

• Loading amount: 20-50 μg of total protein per lane is typically sufficient

Western Blot Protocol:

• Gel percentage: 10-12% SDS-PAGE gels are appropriate for resolving the 45 kDa BHMT protein

• Transfer conditions: 100V for 60-90 minutes in standard Tris-glycine transfer buffer

• Blocking: 5% non-fat dry milk in TBST for 1 hour at room temperature

• Primary antibody incubation:

  • Dilution: 1:1000 to 1:5000 depending on the specific antibody

  • Incubation: Overnight at 4°C with gentle rocking

• Secondary antibody: HRP-conjugated secondary antibody at 1:2000-1:10000 dilution

• Detection: Enhanced chemiluminescence (ECL) system

Expected Results:

• A distinct band at approximately 45 kDa corresponding to BHMT

• Possible detection of BHMT2 by some antibodies that recognize both proteins

• Mouse liver tissue lysate has been successfully used for Western blot validation

Troubleshooting:

• Multiple bands: May indicate splice variants, degradation products, or non-specific binding

• Weak signal: Increase protein loading or antibody concentration, or extend exposure time

• No signal: Verify BHMT expression in the sample type used; consider positive controls

How can BHMT expression be manipulated in experimental systems for functional studies?

Researchers can manipulate BHMT expression through several established methodologies:

1. siRNA-Mediated Knockdown:

• Validated siRNA sequence targeting BHMT: 5′-GUGAAGACAAGCUGGAAAAd(TT)-3′

• Transfection protocol:

  • Seed 4×10^5 primary hepatocytes per well in a 6-well plate in 1.9 mL medium

  • Prepare transfection complexes containing 5 μg of BHMT siRNA in 200 μL

  • Add dropwise onto cells with gentle swirling for even distribution

  • Incubate for 24-48 hours before analysis

  • Include appropriate negative control siRNA without known adverse effects

2. Plasmid-Based Overexpression:

• Cloning strategy:

  • Clone BHMT open reading frame (GenBank accession number U50929) into expression vector pcDNA3.1

  • Confirm correct insertion by sequencing

  • Purify plasmid using plasmid purification systems (e.g., QIAwell)

• Transfection:

  • Transfect hepatic cell lines (e.g., HepG2) using appropriate transfection reagent

  • Select stable transfectants using Zeocin selection

  • Verify expression by Western blot

3. Stable Cell Line Generation:

• The pcDNA3.1-BHMT vector contains the cytomegalovirus promoter for constitutive expression and Zeocin resistance gene for selection

• After transfection, maintain cells in selection medium for 2-3 weeks

• Isolate and expand individual colonies

• Screen for BHMT expression levels

• Cryopreserve early passages of verified clones

4. In vitro Translation:

• For biochemical studies, in vitro translation can be performed using:

  • TNT® coupled rabbit reticulocyte lysate (RRL) system

  • 1 μg of expression construct DNA in 25 μL RRL

  • Addition of T7 buffer, T7 polymerase, amino acid mixture lacking methionine

  • Include labeled methionine (e.g., 35S-methionine) for detection

  • Incubate at 30°C for 90 minutes

  • Analyze by SDS-PAGE and autoradiography

What are the best practices for immunohistochemical detection of BHMT in tissue samples?

For optimal immunohistochemical detection of BHMT in tissue samples, researchers should follow these methodological guidelines:

Sample Preparation:

• Fixation: 10% neutral buffered formalin for 24-48 hours

• Processing: Standard paraffin embedding

• Sectioning: 4-5 μm thickness sections on charged slides

• Target tissues: Liver and kidney are optimal for positive control

Antigen Retrieval:

• Heat-mediated antigen retrieval using Tris-EDTA Buffer (pH 9.0) is recommended

• Pressure cooker or microwave heating methods are effective

• Maintain sections in boiling buffer for 10-20 minutes

• Allow gradual cooling to room temperature

Staining Protocol:

• Deparaffinization and rehydration through xylene and graded alcohols

• Endogenous peroxidase blocking: 3% H₂O₂ for 10 minutes

• Protein blocking: 5-10% normal serum for 30 minutes

• Primary antibody:

  • Dilution range: 1:100 to 1:20,000 depending on the antibody

  • Human kidney tissue samples have been successfully stained at dilutions as high as 1:20,000

  • Incubation: Overnight at 4°C or 1-2 hours at room temperature

• Secondary detection:

  • HRP-conjugated secondary antibody

  • DAB chromogen development: 3-5 minutes with monitoring

• Counterstaining: Hematoxylin for 1-2 minutes

• Dehydration and coverslipping

Expected Results:

• Cytoplasmic staining in human kidney tissue

• Strong staining in hepatocytes

• Variable intensity depending on sample type and fixation

Controls:

• Positive control: Known BHMT-expressing tissue (liver or kidney)

• Negative control: Omission of primary antibody, replaced with buffer

• Secondary antibody only control: PBS instead of primary antibody

Why might Western blot detection of BHMT show inconsistent results, and how can these issues be resolved?

Inconsistent Western blot results for BHMT detection can stem from several methodological factors. Here are common issues and their solutions:

Problem 1: Multiple bands or unexpected band sizes

• Causes:

  • Protein degradation during sample preparation

  • Post-translational modifications

  • Cross-reactivity with BHMT2 (approximate size also ~45 kDa)

  • Splice variants of BHMT

• Solutions:

  • Use fresh samples and maintain low temperature during preparation

  • Add protease inhibitor cocktail to lysis buffer

  • Confirm antibody specificity—some antibodies detect both BHMT and BHMT2

  • For research requiring specific detection, select antibodies validated for distinguishing between BHMT and BHMT2

Problem 2: Weak or absent signal

• Causes:

  • Low BHMT expression in sample

  • Inefficient protein transfer

  • Suboptimal antibody concentration

  • Degraded antibody

• Solutions:

  • Use positive control tissue (liver or kidney) to validate protocol

  • Increase protein loading (40-60 μg)

  • Optimize transfer conditions (time, buffer, membrane type)

  • Titrate antibody concentration

  • Store antibody according to manufacturer's recommendations

  • Some antibodies require specific buffer conditions for optimal performance

Problem 3: High background

• Causes:

  • Insufficient blocking

  • Too high antibody concentration

  • Cross-reactivity with other proteins

• Solutions:

  • Extend blocking time or try alternative blocking reagents

  • Increase washing steps (5x5 minutes with TBST)

  • Titrate antibody to optimal concentration

  • Consider more specific monoclonal antibodies

Problem 4: Inconsistent results between experiments

• Causes:

  • Variable BHMT expression in response to experimental conditions

  • Instability of BHMT protein under certain conditions

• Solutions:

  • Standardize sample collection and processing

  • Document experimental conditions that might affect BHMT expression

  • Consider that homocysteine levels can affect BHMT stability

  • Use housekeeping proteins as loading controls

How can researchers validate the specificity of BHMT antibodies for their particular experimental system?

Validating BHMT antibody specificity is crucial for experimental reliability. Researchers should implement these methodological approaches:

Positive and Negative Control Tissues/Cells

• Positive controls:

  • Human or mouse liver and kidney tissues (known high BHMT expression)

  • Hepatic cell lines (HepG2, Hep3B) with confirmed BHMT expression

• Negative controls:

  • Tissues with minimal BHMT expression

  • Cell lines lacking BHMT expression

  • BHMT knockout models or BHMT-knockdown cells using siRNA approaches

Peptide Competition Assay

• Pre-incubate the BHMT antibody with excess immunizing peptide

• Run parallel assays with blocked and unblocked antibody

• Specific signals should be eliminated or significantly reduced in the peptide-blocked sample

Multiple Antibody Validation

• Use two or more antibodies targeting different epitopes of BHMT

• Concordant results from different antibodies increase confidence in specificity

• Compare monoclonal and polyclonal antibodies for confirmation

Genetic Manipulation Controls

• Overexpression: Transfect cells with BHMT expression constructs

  • Signal should increase proportionally to expression level

• Knockdown: Use BHMT siRNA (e.g., 5′-GUGAAGACAAGCUGGAAAAd(TT)-3′)

  • Signal should decrease proportionally to knockdown efficiency

• These manipulations provide powerful specificity controls

Mass Spectrometry Validation

• Immunoprecipitate BHMT using the antibody

• Confirm protein identity by mass spectrometry

• This provides definitive proof of antibody specificity

Cross-Reactivity Assessment

• Test for cross-reactivity with BHMT2

• Some antibodies are designed to detect both proteins, while others are specific

• Antibodies like EPR20822 recognize both BHMT and BHMT2

• Choose appropriate antibody based on experimental needs

What considerations are important when using BHMT antibodies for co-immunoprecipitation studies investigating protein-protein interactions?

When designing co-immunoprecipitation (co-IP) experiments with BHMT antibodies to investigate protein-protein interactions, researchers should consider these methodological factors:

Antibody Selection for IP

• Consider epitope accessibility:

  • Select antibodies that recognize surface-exposed epitopes in the native conformation

  • Avoid antibodies that target regions involved in protein-protein interactions

• Antibody format:

  • Pre-conjugated antibodies to agarose or magnetic beads minimize non-specific binding

  • For unconjugated antibodies, protein A/G beads can be used

  • Anti-HA immunoprecipitation kits have been successfully used for HA-tagged BHMT2 studies

Lysis Buffer Optimization

• Buffer composition is critical:

  • Mild non-ionic detergents (0.5-1% NP-40 or Triton X-100) preserve protein-protein interactions

  • Avoid harsh detergents (SDS, deoxycholate) that disrupt protein interactions

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation-dependent interactions

• Salt concentration:

  • 150 mM NaCl is standard, but may need optimization

  • Higher salt reduces non-specific binding but may disrupt weak interactions

Experimental Controls

• Essential controls include:

  • Input control: Small aliquot of pre-IP lysate

  • Negative control: Non-specific antibody of same isotype

  • Bead-only control: Beads without antibody

  • Reverse IP: IP with antibody against suspected interaction partner

  • If using tagged proteins, include untagged controls

Special Considerations for BHMT

• BHMT-BHMT2 interaction:

  • Research has shown that BHMT and BHMT2 can co-precipitate

  • BHMT appears to stabilize BHMT2 in expression systems

  • When studying BHMT2 interactions, co-expression with BHMT may be necessary

• Homocysteine effects:

  • Consider that homocysteine can stabilize BHMT

  • This may influence interaction dynamics in experimental systems

Detection Methods

• Western blot analysis:

  • After IP, perform Western blot analysis to detect co-precipitated proteins

  • Use antibodies specific to suspected interaction partners

  • Consider that IP antibodies may interfere with detection—use light chain specific secondary antibodies

• Mass spectrometry:

  • For unbiased discovery of interaction partners

  • Requires careful controls to distinguish true interactors from contaminants

Protocol Example

• Following successful implementation in published research:

  • Transfect cells with constructs for BHMT, HA-tagged BHMT2, or both

  • Lyse cells in appropriate buffer

  • Incubate cell lysates with anti-HA-agarose for 1 hour

  • Wash beads thoroughly

  • Elute bound proteins in SDS sample buffer

  • Perform Western blot analysis to detect co-precipitated proteins

How can researchers accurately quantify BHMT expression levels using antibody-based methods?

Accurate quantification of BHMT expression requires careful methodological consideration and appropriate controls. Here are comprehensive guidelines:

Western Blot Quantification

• Sample preparation standardization:

  • Extract proteins using consistent protocols

  • Determine protein concentration by BCA or Bradford assay

  • Load equal amounts of total protein (20-50 μg)

• Quantification procedure:

  • Include standard curve of recombinant BHMT when absolute quantification is needed

  • Use housekeeping proteins (β-actin, GAPDH) as loading controls

  • Capture images within linear range of detection

  • Use densitometry software for analysis

  • Report results as normalized ratios to control

• Technical considerations:

  • Run biological replicates (n≥3) for statistical validity

  • Include positive control samples (liver/kidney tissue)

  • Perform technical replicates of each sample when possible

ELISA-Based Quantification

• Advantages for BHMT quantification:

  • Higher throughput than Western blotting

  • Greater sensitivity and dynamic range

  • More precise quantification

• Protocol considerations:

  • Capture antibody: Anti-BHMT antibody pre-coated on plate

  • Detection antibody: Secondary anti-BHMT antibody (different epitope)

  • Standard curve: Purified recombinant BHMT protein

  • Sample dilution: Determine optimal dilution to fall within standard curve

  • Multiple washes between steps to reduce background

• Data analysis:

  • Generate standard curve using purified BHMT

  • Calculate BHMT concentration in samples from standard curve

  • Report as ng/mL or normalized to total protein

Immunohistochemistry/Immunofluorescence Quantification

• Semi-quantitative scoring:

  • Develop consistent scoring system (0-3+ or 0-100%)

  • Score independently by multiple observers

  • Include reference images for standardization

• Digital image analysis:

  • Capture images under identical conditions

  • Use software to quantify staining intensity

  • Set consistent thresholds for positive staining

  • Report as mean intensity or percentage of positive area

• Contextual considerations:

  • Document subcellular localization (cytoplasmic/nuclear)

  • Note tissue-specific expression patterns

  • Compare to positive control tissues

Flow Cytometry

• Protocol optimization:

  • Cell fixation and permeabilization for intracellular BHMT

  • Antibody titration to determine optimal concentration

  • Staining at 2-5 μg for 1×10^6 cells has been reported effective

• Controls:

  • Unstained cells for autofluorescence

  • Isotype control for non-specific binding

  • FMO (fluorescence minus one) controls

• Analysis:

  • Report as mean fluorescence intensity (MFI)

  • Calculate fold change relative to control

  • Determine percentage of BHMT-positive cells

RT-qPCR Correlation

• While not antibody-based, correlating protein expression with mRNA levels provides valuable validation:

  • Extract RNA and protein from parallel samples

  • Perform RT-qPCR for BHMT mRNA quantification

  • Compare protein/mRNA ratios across samples

  • Discrepancies may indicate post-transcriptional regulation

Human error, human variation, and bias can significantly impact quantification. Implementing blinded analysis, technical replicates, and automated analysis tools can minimize these effects and improve data reliability.

How are BHMT antibodies being utilized in emerging research areas beyond traditional applications?

BHMT antibodies are finding novel applications in several cutting-edge research areas:

Cancer Biology Research

• Metabolic reprogramming studies:

  • Investigation of one-carbon metabolism alterations in cancer cells

  • Analysis of BHMT expression changes in hepatocellular carcinoma and other cancers

  • Correlation of BHMT levels with tumor progression and patient outcomes

• Methodological approach:

  • Tissue microarray analysis of tumor samples with immunohistochemistry

  • Combined analysis with other metabolic enzymes (MTHFR, MTR)

  • Correlation with clinical parameters and survival data

Neurodegenerative Disease Research

• Homocysteine-related neurodegeneration:

  • Examination of BHMT expression in brain tissues

  • Investigation of BHMT's role in homocysteine regulation and neuronal health

  • Potential connections to Alzheimer's and other neurodegenerative conditions

• Experimental strategies:

  • Comparative BHMT immunostaining in control vs. disease brain tissues

  • Analysis of BHMT in cerebrospinal fluid

  • Cell culture models examining BHMT overexpression effects on neuronal survival

Single-Cell Analysis

• Heterogeneity of BHMT expression:

  • Single-cell protein analysis in liver and kidney tissues

  • Identification of BHMT-expressing subpopulations

  • Correlation with cellular metabolic states

• Technical approaches:

  • Mass cytometry (CyTOF) incorporating BHMT antibodies

  • Imaging mass cytometry for spatial distribution analysis

  • Single-cell Western blotting techniques

Multiplexed Imaging

• Spatial context of BHMT expression:

  • Co-localization with metabolic enzymes and regulatory proteins

  • Tissue microenvironment influence on BHMT expression

• Advanced techniques:

  • Multiplex immunofluorescence with tyramide signal amplification

  • Imaging mass cytometry for high-parameter tissue analysis

  • Cyclic immunofluorescence for extended biomarker panels

Liquid Biopsy Development

• Circulating BHMT as biomarker:

  • Detection of BHMT in serum/plasma from liver disease patients

  • Correlation with tissue damage and disease progression

• Methodological considerations:

  • Highly sensitive ELISA development using optimized antibody pairs

  • Digital ELISA (Simoa) approaches for ultrasensitive detection

  • Correlation with established liver damage markers