mdh1b Antibody

What is MDH1B and what is its function in human cells?

MDH1B (Malate Dehydrogenase 1B, NAD Soluble) is part of the malate dehydrogenase superfamily that has been detected at the transcript level in humans . It is predicted to be involved in NADH metabolic processes, dicarboxylic acid metabolic pathways, and the tricarboxylic acid cycle . While MDH1B's precise function remains under investigation, it shares structural similarities with other malate dehydrogenases that catalyze the reversible conversion of malate to oxaloacetate using NAD+ as a cofactor.

What are the common applications for MDH1B antibodies in research?

MDH1B antibodies are primarily utilized in Western Blotting (WB) and immunohistochemistry (IHC) applications to detect and quantify MDH1B protein expression in human tissues and cell lines . These antibodies are also employed in ELISA assays for quantitative analysis of MDH1B in biological samples including serum, plasma, cell culture supernatants, and other biological fluids . Emerging applications include their use in mechanistic studies investigating metabolic pathways and potential roles in cancer metabolism.

What epitopes of MDH1B are commonly targeted by commercial antibodies?

Commercial MDH1B antibodies typically target several regions of the protein:

• Central region antibodies targeting amino acids 304-332

• C-terminal antibodies

• Full-length antibodies targeting amino acids 1-518

The choice of epitope can significantly impact specificity and application suitability, with central region antibodies (304-332) being particularly common for human MDH1B detection .

What are the recommended protocols for using MDH1B antibodies in Western blotting?

For optimal Western blotting results with MDH1B antibodies:

• Sample preparation: Extract proteins using standard lysis buffers containing protease inhibitors

• Gel electrophoresis: Load 30-35μg of protein per lane on 5-20% SDS-PAGE gels

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

• Blocking: Block membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: Dilute MDH1B antibody 1:1000 and incubate overnight at 4°C

• Washing: Wash membrane with TBS-0.1% Tween three times (5 minutes each)

• Secondary antibody: Incubate with appropriate HRP-conjugated secondary antibody (1:500) for 1.5 hours at room temperature

• Detection: Develop using enhanced chemiluminescence (ECL) detection system

The expected band size for MDH1B is approximately 58 kDa .

How should MDH1B antibodies be optimized for immunohistochemistry?

For IHC applications with MDH1B antibodies:

• Tissue preparation: Use paraffin-embedded sections of human tissue

• Antigen retrieval: Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Blocking: Block tissue sections with 10% goat serum to minimize background

• Primary antibody: Dilute MDH1B antibody 1:1000-1:2500 and incubate overnight at 4°C

• Secondary antibody: Use peroxidase-conjugated anti-rabbit IgG as secondary antibody and incubate for 30 minutes at 37°C

• Detection: Develop using DAB as the chromogen

Optimal dilution should be determined empirically for each tissue type and fixation method.

What are the considerations when using MDH1B ELISA kits?

When implementing MDH1B ELISA assays:

• Sample types: Compatible with serum, plasma, cell culture supernatants, and other biological fluids

• Assay principle: The sandwich ELISA format employs a pre-coated antibody specific for MDH1B

• Protocol overview:

  • Samples and standards are pipetted into wells where MDH1B binds to immobilized antibody

  • After washing, a biotin-conjugated antibody specific for MDH1B is added

  • Following washing, streptavidin-HRP is added to wells

  • After a final wash, substrate solution is added, producing color proportional to MDH1B amount

• Duration: Multiple-step standard sandwich ELISA with working time of 3-5 hours

• Specificity: Assays show high specificity with no significant cross-reactivity with analogues

How can MDH1B antibodies be used to study metabolic reprogramming in cancer?

MDH1B antibodies can provide valuable insights into cancer metabolic reprogramming through:

• Expression analysis: Quantifying MDH1B expression changes across different cancer types and stages

• Localization studies: Using fluorescently conjugated MDH1B antibodies (FITC, PE) to track subcellular localization changes during cancer progression

• Metabolic pathway investigation: Correlating MDH1B expression with HIF-1α and other metabolic markers

• Therapeutic targeting validation: Monitoring effects of MDH1/2 dual inhibitors on MDH1B expression and metabolic profiles

Research has shown that dual MDH1/2 inhibitors can reduce ATP content in lung cancer cells and suppress HIF-1α accumulation, potentially providing new therapeutic avenues .

What are the technical considerations when using conjugated MDH1B antibodies?

When working with conjugated MDH1B antibodies (PE, FITC, HRP, etc.):

• Signal-to-noise optimization:

  • PE-conjugated antibodies: Optimal for flow cytometry with higher sensitivity than FITC

  • FITC-conjugated antibodies: Suitable for immunofluorescence but more susceptible to photobleaching

  • HRP-conjugated antibodies: Preferred for enhanced sensitivity in Western blotting and ELISA

• Handling considerations:

  • Protect fluorophore-conjugated antibodies from light during all steps

  • Store at recommended temperatures (typically 4°C short-term, -20°C long-term)

  • Avoid repeated freeze-thaw cycles that can degrade conjugates

• Application-specific dilutions:

  • Western blotting: 1:1000 for HRP-conjugated MDH1B antibodies

  • ELISA: 1:1000-1:2000 for most conjugated formats

  • Flow cytometry: 1:200-1:500 for PE or FITC conjugates

What approaches can resolve cross-reactivity issues with MDH1B antibodies?

To address potential cross-reactivity with other malate dehydrogenase family members:

• Epitope selection strategy:

  • Choose antibodies targeting the central region (AA 304-332) where MDH1B sequence diverges from MDH1 and MDH2

  • Validate specificity using knockout/knockdown controls

  • Consider peptide competition assays to confirm binding specificity

• Experimental validation:

  • Run parallel reactions with MDH1, MDH2, and MDH1B recombinant proteins

  • Include neutralizing peptides corresponding to the immunogen sequence

  • Implement dual-staining approaches to distinguish between isoforms

• Data interpretation:

  • Compare molecular weights (MDH1B: ~58kDa vs. MDH1: ~36kDa)

  • Validate with orthogonal detection methods (mass spectrometry)

  • Analyze subcellular fractionation results to distinguish between cytosolic and mitochondrial isoforms

How should researchers interpret MDH1B expression patterns across different tissues?

When analyzing MDH1B expression patterns:

• Baseline expression assessment:

  • Compare expression levels in normal tissues against established housekeeping proteins

  • Consider tissue-specific metabolic requirements when interpreting expression levels

  • Note that MDH1B expression may vary significantly between tissues based on metabolic activity

• Comparative analysis framework:

  • Implement quantitative densitometry for Western blot data normalization

  • Use appropriate statistical methods for comparing multiple tissue types

  • Consider both protein and transcript levels for comprehensive analysis

• Physiological context interpretation:

  • Correlate MDH1B expression with other TCA cycle enzymes

  • Assess relationships with NADH/NAD+ ratios in different tissues

  • Interpret expression in context of tissue-specific metabolic demands

What controls should be included when validating MDH1B antibody specificity?

A comprehensive validation approach includes:

How can researchers integrate MDH1B antibody data with other omics approaches?

For comprehensive multi-omics integration:

• Correlation with transcriptomics:

  • Compare protein levels detected by MDH1B antibodies with mRNA expression data

  • Investigate potential post-transcriptional regulation mechanisms when discrepancies occur

  • Use transcript data to guide tissue selection for antibody-based studies

• Integration with metabolomics:

  • Correlate MDH1B protein levels with malate, oxaloacetate, and NADH/NAD+ ratios

  • Investigate metabolic flux through MDH1B by combining protein expression with isotope labeling studies

  • Assess impact of MDH1B inhibition on broader metabolome profiles

• Functional genomics connections:

  • Combine antibody-based protein quantification with genetic perturbation screens

  • Implement active learning approaches for predicting MDH1B-substrate interactions

  • Develop machine learning models that incorporate antibody-derived protein data with genetic information to predict metabolic phenotypes

How are MDH1B antibodies being used to study cancer metabolism?

MDH1B antibodies are increasingly valuable in cancer metabolism research:

• Metabolic rewiring assessment:

  • Quantify MDH1B expression changes across cancer progression stages

  • Correlate with hypoxia markers to understand metabolic adaptation

  • Investigate associations with glycolysis versus oxidative phosphorylation balance

• Therapeutic response monitoring:

  • Track MDH1B expression changes following treatment with metabolism-targeting drugs

  • Assess effects of dual MDH1/2 inhibitors on cancer cell bioenergetics

  • Correlate inhibition efficiency with anti-tumor effects

• Translational applications:

  • Develop prognostic and predictive biomarker panels including MDH1B

  • Investigate potential for therapeutic antibodies targeting MDH1B

  • Assess MDH1B as part of metabolic vulnerability signatures in precision oncology

What are the methodological approaches for studying MDH1B in the context of the NAD+/NADH balance?

When investigating MDH1B's role in NAD+/NADH homeostasis:

• Direct enzymatic activity assessment:

  • Measure MDH1B activity using recombinant protein and NAD+/NADH assays

  • Compare with mitochondrial and cytosolic MDH isoforms

  • Determine substrate preferences and kinetic parameters

• Cellular redox state correlation:

  • Use genetically encoded NAD+/NADH sensors alongside MDH1B immunostaining

  • Perform subcellular fractionation to determine compartment-specific effects

  • Implement metabolic flux analysis with stable isotope tracers

• Genetic perturbation approaches:

  • Create MDH1B knockdown/knockout cell models

  • Assess compensatory changes in other MDH isoforms

  • Measure effects on cellular NAD+/NADH ratios and dependent processes