LDHB Antibody

What is LDHB and what cellular functions does it serve in experimental models?

LDHB, also known as LDH-H and NY-REN-46, belongs to the LDH/MDH superfamily and catalyzes the reversible conversion of lactate and pyruvate with NAD+ as a cofactor. Specifically, LDHB catalyzes the reaction: (S)-lactate + NAD+ = pyruvate + NADH . In research contexts, LDHB is studied for its role in cellular metabolism, particularly in cells that rely on lactate metabolism.

In human β cells, LDHB acts to limit lactate levels, prevent inappropriate insulin release at low glucose concentration, and shape glucose-stimulated insulin secretion . This makes it an important target for diabetes and metabolic research. The protein has a calculated molecular weight of approximately 36-37 kDa, though it is commonly observed at 35 kDa in experimental contexts .

What are the validated applications for LDHB antibodies in research protocols?

LDHB antibodies have been validated for multiple experimental applications across different research contexts:

When designing experiments, it is recommended to optimize antibody concentration for each specific application and sample type. For most commercially available antibodies, tissue-specific validations have been performed in multiple cell lines including HeLa, DU 145, HEK-293T, Jurkat, and PC-3 cells, as well as mouse/rat heart tissue .

What methodological considerations should researchers account for when performing immunohistochemistry with LDHB antibodies?

When performing IHC with LDHB antibodies, several methodological considerations are critical:

• Antigen retrieval method: TE buffer pH 9.0 is typically recommended, though citrate buffer pH 6.0 can serve as an alternative . The choice of retrieval method can significantly impact staining intensity and specificity.

• Antibody dilution range: A starting dilution of 1:300-1:1200 is recommended for IHC applications , with some antibodies performing optimally at 1:400-1:1500 .

• Tissue-specific considerations: LDHB antibodies have been validated on human ovary cancer tissue , human kidney tissue, and human prostate cancer tissue . Different tissues may require protocol adjustments.

• Expected localization pattern: LDHB typically shows cytoplasmic localization throughout β cells, with differential expression patterns in α cells (approximately 19% of α cells express high levels of LDHB) .

• Controls: Include both positive tissue controls and negative controls (omitting primary antibody) to ensure specificity.

How does LDHB expression correlate with sensitivity to therapeutic antibodies in cancer research models?

LDHB has been identified as a potential predictive marker for sensitivity to anti-EGFR monoclonal antibodies (mAbs) in colorectal cancer (CRC) cell lines. Comprehensive proteome analysis revealed that LDHB expression levels were significantly upregulated with the acquisition of resistance to cetuximab in previously cetuximab-sensitive CRC cell lines .

Specifically, cytoplasmic LDHB was detected as a marker of cetuximab sensitivity, with increased expression in cetuximab-resistant CRC cell lines . This finding suggests that LDHB expression monitoring could potentially serve as a biomarker for predicting treatment response.

For researchers investigating drug resistance mechanisms, monitoring changes in LDHB expression before and after treatment may provide insights into resistance development pathways. Methodologically, both proteomics approaches and antibody-based detection methods can be employed to track these changes in experimental models.

What mechanisms link LDHB inhibition to mitochondrial dynamics and mitophagy in cellular models?

Research utilizing LDHB inhibition via specific siRNA has revealed significant effects on mitochondrial morphology and function. Key findings include:

• Mitochondrial morphology changes: TEM images showed elliptic mitochondria with dramatic loss of mitochondrial cristae in LDHB-inhibited cells compared to controls .

• Mitophagy induction: LDHB inhibition resulted in increased numbers of mitochondria trapped by double or single membrane vesicles, with quantitative analysis showing a significant increase in autophagosome-like structures .

• Mitochondrial fragmentation: Laser confocal microscopy using TOMM20 antibody labeling demonstrated shorter mitochondrial length and increased division upon LDHB inhibition .

• Protein expression changes: Expression of mitophagy proteins TOMM20 and VDAC1 decreased upon LDHB inhibition, while ubiquitination of MFN2 (a mitochondrial fusion mediator) increased .

For researchers investigating mitochondrial dynamics, these findings suggest LDHB as a potential regulatory target. Methodologically, dual fluorescence reporters (such as mito-mRFP-EGFP) can be utilized to analyze the delivery of autophagosomes to lysosomes in LDHB-inhibited cells .

How can researchers effectively validate LDHB antibody specificity in experimental systems?

Antibody validation is critical for ensuring experimental reproducibility. For LDHB antibodies, several validation approaches have proven effective:

• Genetic knockdown validation: siRNA-mediated knockdown of LDHB should result in a corresponding reduction in antibody signal. For example, a 3-fold reduction in LDHB expression was observed in EndoC-βH1 cells treated with siRNA against LDHB compared to control .

• Knockout cell lines: Several publications have validated LDHB antibodies in knockout/knockdown systems .

• Cross-reactivity testing: Test antibodies on multiple species to confirm predicted reactivity. Commercial LDHB antibodies have been validated for human, mouse, and rat samples, with some showing extended reactivity to pig, chicken, bovine, and sheep models .

• Multiple antibody comparison: Using different antibodies targeting distinct epitopes of LDHB can provide confirmation of specificity.

• Peptide competition: Pre-incubation with the immunizing peptide should abolish specific staining in appropriate applications.

What is the relationship between LDHB and viral infection mechanisms in host cells?

LDHB has been implicated in viral infection mechanisms, particularly in relation to the Classical Swine Fever Virus (CSFV). Key research findings include:

• Protein-protein interactions: LDHB interacts with the NS3 protein of CSFV, as demonstrated through co-immunoprecipitation and confocal microscopy showing colocalization in the cytoplasm .

• Reciprocal regulation: LDHB inhibits the expression of NS3, and NS3 also inhibits the expression of LDHB in CSFV-infected PK-15 and 3D4/2 cells .

• Mitophagy regulation: LDHB inhibition induces mitochondrial fission and mitophagy, which also inhibits the activation of NFKB signaling and apoptosis when co-infected with CSFV .

• Viral replication impact: LDHB inhibition promotes CSFV growth via mitophagy, whereas its overexpression decreased CSFV replication .

For virus-host interaction research, these findings suggest that tracking LDHB levels and activity could provide insights into mechanisms of viral persistence. Methodologically, researchers investigating these interactions should consider dual immunofluorescence approaches to visualize LDHB-viral protein colocalization.

How does LDHB expression and distribution differ between pancreatic cell types and under metabolic stress conditions?

LDHB shows distinct expression patterns across pancreatic cell types with significant changes under metabolic stress:

For researchers investigating pancreatic biology and diabetes, these findings highlight the importance of considering cellular heterogeneity when studying metabolic enzymes. Methodologically, immunofluorescence co-staining with cell-type specific markers provides the most informative approach to analyzing LDHB distribution.

What are common issues encountered with LDHB antibodies and how can researchers address them?

When working with LDHB antibodies, researchers may encounter several technical challenges:

• Variable background staining: Optimize blocking conditions (typically 5% BSA or normal serum from the secondary antibody species) and consider longer blocking times (1-2 hours at room temperature).

• Cross-reactivity concerns: LDHB has high sequence homology with other LDH family members. Validate specificity through knockdown experiments or using recombinant protein controls.

• Inconsistent band sizes: While the calculated molecular weight of LDHB is approximately 36-37 kDa, it is commonly observed at 35 kDa in experimental contexts . Post-translational modifications may affect migration patterns.

• Tissue-specific variations: LDHB expression varies significantly between tissues and cell types. Expression is generally higher in human compared to mouse islets, with slightly higher levels in exocrine versus endocrine compartments .

• Species cross-reactivity limitations: Always verify species reactivity claims with pilot experiments when working with less common model organisms.

What are the optimal sample preparation methods for different LDHB antibody applications?

Sample preparation significantly impacts LDHB antibody performance across applications:

For Western Blot:

• Optimal lysis buffers typically contain 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris pH 8.0, with protease inhibitors

• Samples should be denatured at 95°C for 5 minutes in Laemmli buffer containing SDS and β-mercaptoethanol

• Loading 20-50 μg of total protein per lane is typically sufficient for detection

For Immunohistochemistry:

• Formalin-fixed, paraffin-embedded (FFPE) tissue sections (4-6 μm) are commonly used

• Antigen retrieval with TE buffer pH 9.0 is recommended, with citrate buffer pH 6.0 as an alternative

• For fresh frozen sections, acetone or methanol fixation may provide better epitope preservation

For Immunofluorescence/ICC:

• Cells should be fixed with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes is typically sufficient

• Blocking with 5% normal serum or BSA for 30-60 minutes reduces non-specific binding

For Immunoprecipitation:

• Non-denaturing lysis buffers containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris pH 7.5, with protease inhibitors

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Pre-clearing lysates with Protein A/G beads can reduce non-specific binding

What emerging applications of LDHB antibodies hold promise for translational research?

Several emerging applications of LDHB antibodies show potential for translational research:

• Predictive biomarker development: The identification of LDHB as a potential marker for anti-EGFR monoclonal antibody sensitivity in colorectal cancer suggests applications in personalized medicine approaches . LDHB antibodies could be developed into companion diagnostic tools.

• Metabolic disease mechanisms: LDHB's role in regulating lactate levels and insulin secretion in β cells points to potential applications in diabetes research and therapeutic development .

• Mitochondrial dynamics and disease: The link between LDHB inhibition and mitophagy suggests applications in studying diseases associated with mitochondrial dysfunction, including neurodegenerative disorders and certain cancers .

• Viral-host interactions: LDHB's involvement in viral infection mechanisms, particularly its interaction with viral proteins and impact on viral replication, opens avenues for antiviral research strategies .

• Tissue heterogeneity analysis: The differential expression of LDHB in pancreatic cell subtypes suggests applications in studying cellular heterogeneity and functional specialization .

For researchers pursuing these directions, combining LDHB antibody-based approaches with emerging technologies like spatial transcriptomics and single-cell proteomics may provide deeper insights into LDHB's context-specific functions.

What are the current limitations in LDHB antibody technology that require further development?

Despite their utility, current LDHB antibody technologies face several limitations requiring further development:

• Isoform specificity: Better tools are needed to distinguish between different LDH family members in complex tissues where multiple isoforms are expressed.

• Post-translational modification detection: Antibodies specifically recognizing phosphorylated, acetylated, or otherwise modified LDHB would enhance functional studies.

• Quantitative applications: Improved standardization for quantitative applications, particularly for comparing LDHB levels across different experimental conditions.

• Live-cell imaging compatibility: Development of non-interfering antibody fragments or nanobodies that can track LDHB in living cells without disrupting function.

• Multiplexing capacity: Enhanced compatibility with multiplexed imaging techniques to simultaneously visualize LDHB alongside multiple other markers.

Addressing these limitations will require continued antibody engineering efforts and validation across diverse experimental systems and disease models.