BDH1 Antibody

What is BDH1 and what is its biological function?

BDH1 (3-hydroxybutyrate dehydrogenase, type 1) is a 343 amino acid protein that localizes to the mitochondrial matrix and belongs to the short-chain dehydrogenases/reductases (SDR) family. The mature protein contains a 46-residue leader peptide for mitochondrial targeting . BDH1 plays a crucial role in ketone body metabolism, specifically in the conversion of β-hydroxybutyrate (βOHB) to acetoacetate (AcAc) in the mitochondria. This metabolic pathway is particularly important during periods of fasting or ketogenic diet adaptation, where ketone bodies serve as alternative energy sources . Recent research has demonstrated that BDH1-mediated βOHB metabolism is protective against reactive oxygen species (ROS) overproduction through the activation of Nrf2 signaling pathways .

What applications are BDH1 antibodies suitable for?

BDH1 antibodies have been validated for multiple experimental applications including:

It is recommended that researchers titrate the antibody in each testing system to obtain optimal results, as optimal dilutions can be sample-dependent . BDH1 antibodies have been successfully used in knockout/knockdown validation studies, with multiple publications confirming their specificity and utility .

Which species reactivity has been validated for BDH1 antibodies?

The following table summarizes the species reactivity data for BDH1 antibodies:

When selecting a BDH1 antibody for your research, ensure the antibody has been validated for your species of interest .

What is the optimal protocol for using BDH1 antibodies in Western Blot analyses?

For Western Blot applications, BDH1 antibodies should be used at dilutions ranging from 1:2000 to 1:10000 depending on your sample type and detection system . The expected molecular weight of BDH1 is 38 kDa (calculated), though the observed molecular weight in SDS-PAGE is typically around 31 kDa . This discrepancy may be due to post-translational modifications or protein processing.

When performing Western Blot with BDH1 antibodies:

• Ensure complete protein denaturation with appropriate sample buffer containing SDS and reducing agents

• Use freshly prepared samples when possible, as BDH1 may degrade during long-term storage

• Positive controls should include tissues with known high BDH1 expression such as liver tissues, HSC-T6 cells, HepG2 cells, HT-29 cells, or COLO 320 cells

• Block with 5% non-fat milk or BSA in TBST for at least 1 hour at room temperature

• Incubate with primary antibody at 4°C overnight followed by appropriate secondary antibody

Potential technical challenges include non-specific binding, which can be addressed by optimizing blocking conditions and antibody dilutions.

How should BDH1 antibodies be used for immunohistochemistry (IHC) staining?

For IHC applications, the recommended dilution range for BDH1 antibodies is 1:500-1:2000 . Antigen retrieval is a critical step for successful IHC with BDH1 antibodies. The search results suggest using TE buffer pH 9.0 for antigen retrieval, though citrate buffer pH 6.0 may also be effective as an alternative .

The detailed protocol for IHC with BDH1 antibodies should include:

• Deparaffinization and rehydration of tissue sections

• Antigen retrieval using TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0

• Blocking of endogenous peroxidase activity with hydrogen peroxide

• Protein blocking with serum or BSA

• Incubation with primary BDH1 antibody at the appropriate dilution

• Detection with a suitable secondary antibody and chromogen

• Counterstaining, dehydration, and mounting

Human liver cancer tissue has been validated as a positive control for BDH1 IHC staining .

What are the proper storage conditions for maintaining BDH1 antibody activity?

BDH1 antibodies should be stored at -20°C for maximum stability. The formulation typically includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, the antibody remains stable for one year after shipment. For small volume antibodies (20μl sizes), the formulation may contain 0.1% BSA as a stabilizer .

Important storage considerations:

• Aliquoting is generally unnecessary for -20°C storage

• Avoid repeated freeze-thaw cycles which can degrade antibody performance

• When removing from storage, thaw completely before use and mix gently to ensure homogeneity

• Keep the antibody on ice when in use but avoid prolonged exposure

How can BDH1 antibodies be used to study metabolic disorders and liver diseases?

BDH1 antibodies are valuable tools for investigating metabolic dysfunction-associated fatty liver disease (MAFLD) and related metabolic disorders. Research has demonstrated that BDH1 expression is downregulated in fatty liver, and this reduction correlates with increased ROS production, inflammation, and apoptosis .

In experimental settings, BDH1 antibodies can be used to:

• Assess BDH1 expression levels in liver tissue samples from patients with metabolic disorders

• Monitor BDH1 expression changes in response to therapeutic interventions

• Validate BDH1 knockdown or overexpression in experimental models

• Co-localize BDH1 with other mitochondrial markers to assess mitochondrial function

A recent study successfully used adeno-associated virus (AAV)-mediated BDH1 overexpression to reverse hepatic function indexes, fibrosis, inflammation, and apoptosis in fatty livers from db/db mice . This approach could represent a novel therapeutic strategy, and BDH1 antibodies play a crucial role in validating the efficacy of such interventions.

What methodologies are available for measuring BDH1 activity in mitochondrial preparations?

BDH1 activity can be measured through a specialized assay using either freshly isolated or frozen mitochondria. The methodology involves:

BDH1 Activity Assay Protocol:

• Use 10μg of mitochondrial preparation

• Prepare assay buffer consisting of Buffer D supplemented with:

  • Alamethicin (0.03mg/ml)

  • Rotenone (0.005mM)

  • NAD+ (2mM)

• Add 200μl of buffer to an opaque 96-well plate

• Add mitochondria followed by 400mM R-3-BHB acid

• Measure NADH production from BDH1 by auto-fluorescence (Ex:Em 340/450nm)

• Convert fluorescence values to pmoles of NADH using an NADH standard curve

This assay provides a functional readout of BDH1 enzymatic activity, complementing expression data obtained through antibody-based techniques. When analyzing BDH1 function in disease models, combining activity assays with expression analysis provides a more comprehensive understanding of how BDH1 dysfunction contributes to pathogenesis.

How can researchers distinguish between BDH1 expression changes and BDH1 activity alterations in disease models?

This is a critical distinction in BDH1 research, as protein expression levels may not always correlate directly with enzymatic activity. To comprehensively assess BDH1 status:

• For expression analysis:

  • Western blot using BDH1 antibodies to quantify total protein levels

  • IHC or IF to assess cellular and subcellular localization

  • qRT-PCR to measure mRNA expression levels

• For activity assessment:

  • BDH1 enzyme activity assay measuring NADH production

  • Metabolomics analysis of BDH1 substrates and products (β-hydroxybutyrate and acetoacetate)

  • Functional mitochondrial assays including respiratory control, membrane potential, and NAD(P)H/NAD(P)+ redox state

Disease models often show discrepancies between expression and activity, possibly due to post-translational modifications, changes in cofactor availability, or alterations in the mitochondrial environment. For instance, in diabetic kidney disease models, BDH1 expression is reduced under high glucose or palmitic acid conditions, but the functional consequences extend beyond simple protein reduction to include ROS overproduction and inflammation .

How does BDH1 function relate to ROS production and antioxidant defense mechanisms?

BDH1-mediated β-hydroxybutyrate (βOHB) metabolism plays a crucial role in cellular antioxidant defense mechanisms. Research shows that:

• BDH1 deficiency promotes ROS overproduction in various cell types, contributing to cellular damage

• This occurs through a pathway involving βOHB-AcAc-succinate-fumarate metabolic flux

• BDH1 knockdown increases ROS levels and reduces mitochondrial membrane potential

• The increased ROS from BDH1 deficiency is primarily of mitochondrial origin

• Treatment with the ROS inhibitor NAC can reverse BDH1 knockdown-mediated increases in apoptosis and inflammatory markers

Conversely, BDH1 overexpression provides protection against ROS-induced damage by:

• Reversing palmitic acid (PA)-induced ROS overproduction

• Preventing mitochondrial dysfunction

• Reducing inflammatory marker activation (IL-1β and IL-18)

• Decreasing apoptotic signaling through caspase-3

These findings position BDH1 as a key regulator of oxidative stress in metabolic disorders and suggest that targeting BDH1 expression or activity could have therapeutic potential.

What role does BDH1 play in diabetic kidney disease, and how can BDH1 antibodies contribute to this research?

BDH1 has emerged as an important factor in diabetic kidney disease (DKD) pathogenesis. Studies show that BDH1 expression is significantly reduced in both diabetic mouse models and in human kidney tissues from diabetic patients . In vitro studies demonstrated that high glucose (HG) or palmitic acid (PA) treatment reduces BDH1 expression in human kidney cells (HK-2) .

Mechanistically, BDH1 deficiency in kidney cells:

• Increases cellular ROS levels

• Elevates pro-inflammatory factors like cleaved IL-1β

• Increases secretion of IL-1β and IL-18

• Contributes to cellular injury through loss of anti-ROS function

BDH1 antibodies are essential tools in this research field for:

• Assessing BDH1 expression in kidney tissue samples from diabetic patients

• Validating BDH1 knockdown or overexpression in experimental models

• Monitoring changes in BDH1 expression in response to therapeutic interventions

• Investigating the subcellular localization of BDH1 in kidney cells

Importantly, adeno-associated virus 9-mediated BDH1 renal expression has shown promise in reversing fibrosis, inflammation, and apoptosis in diabetic kidney models, suggesting potential therapeutic applications .

What contradictions or unexpected findings have emerged in BDH1 research that require further investigation?

Several intriguing contradictions and unexpected findings have emerged in BDH1 research:

• Discrepancy in molecular weight: The calculated molecular weight of BDH1 is 38 kDa, but the observed molecular weight in Western blot is typically around 31 kDa . This discrepancy may reflect post-translational modifications, proteolytic processing, or alternative splicing, but requires further investigation.

• Tissue-specific effects: While BDH1 overexpression ameliorates liver injury in metabolic dysfunction-associated fatty liver disease (MAFLD) models, its effects may differ in other tissues. The mechanisms underlying these tissue-specific effects remain unclear.

• Regulatory mechanisms: The factors governing BDH1 expression in different disease states are not fully understood. For instance, both hyperglycemia and hyperlipidemia reduce BDH1 expression, but the molecular pathways mediating this reduction need further elucidation .

• Therapeutic potential versus physiological role: While BDH1 overexpression shows therapeutic potential in multiple disease models, the physiological significance of BDH1 downregulation in these conditions remains unclear—is it an adaptive response or a pathological change?

• Relationship with ketone metabolism: Although BDH1 is known for its role in ketone metabolism, its protective effects appear to extend beyond this canonical function, suggesting additional mechanisms that warrant investigation.

What are the advantages and limitations of using ELISA for BDH1 quantification?

Advantages of BDH1 ELISA:

• High sensitivity and specificity: BDH1 ELISA kits demonstrate excellent specificity with minimal cross-reactivity with analogues .

• Quantitative results: ELISA provides precise quantification of BDH1 levels, with standard curves typically covering a range suitable for biological samples.

• Sample compatibility: BDH1 ELISA has been validated for multiple sample types:

This high recovery rate across different matrices indicates reliable performance across sample types .

• Higher throughput: ELISA allows for processing multiple samples simultaneously in a 96-well format.

Limitations of BDH1 ELISA:

• Limited information on protein modifications: Unlike Western blotting, ELISA cannot distinguish between different forms of the protein (e.g., phosphorylated vs. non-phosphorylated).

• No information on molecular weight: ELISA cannot confirm the molecular weight of the detected protein.

• Potential cross-reactivity: Despite high specificity, the possibility of cross-reaction with unknown analogues cannot be completely ruled out .

• Sample preparation requirements: Proper sample preparation is critical for accurate results, and matrix effects can influence outcomes.

How can researchers validate BDH1 antibody specificity in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For BDH1 antibodies, researchers should consider these validation approaches:

• Positive and negative controls:

  • Use tissues with known high BDH1 expression (liver tissue, HSC-T6, HepG2, HT-29, or COLO 320 cells) as positive controls

  • Include samples from BDH1 knockout or knockdown models as negative controls

• Knockout/knockdown validation:

  • Utilize siRNA-mediated knockdown of BDH1 to confirm antibody specificity

  • Several publications have used this approach for BDH1 antibody validation

• Overexpression controls:

  • Express tagged BDH1 (e.g., FLAG-tagged) and perform dual detection with tag-specific antibodies and BDH1 antibodies

  • Confirm co-localization and signal intensity correlation

• Western blot analysis:

  • Verify that the detected band appears at the expected molecular weight (approximately 31 kDa)

  • Perform peptide competition assays where available

• Cross-technique validation:

  • Compare results across multiple techniques (WB, IHC, IF, ELISA)

  • Consistent findings across different methods increase confidence in antibody specificity

• Immunoprecipitation followed by mass spectrometry:

  • Use the antibody for immunoprecipitation followed by mass spectrometry to confirm BDH1 identity

What are the most effective methods for measuring BDH1 enzymatic activity in tissue samples?

Measuring BDH1 enzymatic activity provides functional insights beyond expression levels. The most effective methods include:

• Spectrophotometric/fluorometric assays:

  • The most common approach utilizes NAD+ as a cofactor and measures NADH production

  • In the standard protocol, 10μg of mitochondria are incubated with R-3-BHB acid and NAD+

  • NADH production is measured by auto-fluorescence (Ex:Em 340/450nm)

  • Fluorescence values are converted to pmoles of NADH using a standard curve

• Mass spectrometry-based metabolomics:

  • Measures substrates and products of BDH1 activity (β-hydroxybutyrate and acetoacetate)

  • Can be performed on tissue extracts or plasma

  • Provides insights into the metabolic flux through the BDH1 pathway

  • Acylcarnitines, amino acids, and organic acids can be simultaneously measured

• Combined approaches:

  • Mitochondrial isolation followed by BDH1 activity assay

  • Assessment of mitochondrial respiratory control, membrane potential, and NAD(P)H/NAD(P)+ redox state in conjunction with BDH1 activity

  • These combined approaches provide a more comprehensive picture of mitochondrial function related to BDH1 activity

When measuring BDH1 activity, it's important to consider factors that might affect enzymatic function, such as sample handling, storage conditions, and the presence of inhibitors or activators in the experimental system.