LKR/SDH Antibody

What is LKR/SDH and why is it important in biological research?

LKR/SDH (lysine-ketoglutarate reductase/saccharopine dehydrogenase) is a bifunctional enzyme that catalyzes the first two steps in lysine catabolism. The LKR domain combines lysine and α-ketoglutarate to form saccharopine, while the SDH domain converts saccharopine into α-aminoadipic semi-aldehyde and glutamate . This bifunctional enzyme is encoded by a single gene in both plants and animals . LKR/SDH is critically important in research because it regulates lysine homeostasis, influencing nitrogen and carbon balance in organisms . In humans, LKR/SDH is a synonym of the AASS gene (aminoadipate-semialdehyde synthase) and plays roles in transcriptional regulation and other biological processes .

What are the structural characteristics of LKR/SDH protein?

The human LKR/SDH protein has a canonical amino acid length of 926 residues and a molecular weight of approximately 102.1 kilodaltons . The protein is primarily localized in the mitochondria and is notably expressed in multiple tissues including the appendix and duodenum . LKR/SDH belongs to both the AlaDH/PNT protein family and the Saccharopine dehydrogenase protein family . The protein contains two distinct enzymatic domains (LKR and SDH) linked on a single polypeptide chain, with each domain maintaining its respective catalytic activity .

How do LKR/SDH antibodies contribute to understanding lysine metabolism?

LKR/SDH antibodies enable researchers to detect, quantify, and track the LKR/SDH protein in various biological samples and experimental conditions . These antibodies are essential tools for investigating the expression patterns, subcellular localization, and post-translational modifications of LKR/SDH. By using specific antibodies, researchers can determine the abundance of LKR/SDH in different tissues, developmental stages, or in response to various stimuli. For example, monoclonal antibodies specifically recognizing the LKR domain have been used to demonstrate the absence of LKR/SDH polypeptide in knockout mutants and reduced expression in heterozygous plants .

What factors should researchers consider when selecting an LKR/SDH antibody for their experiments?

When selecting an LKR/SDH antibody, researchers should consider several critical factors:

• Target species specificity: Ensure the antibody recognizes the LKR/SDH protein in your experimental organism. Available antibodies include those reactive to human, Arabidopsis, and other species .

• Domain specificity: Determine whether you need an antibody that recognizes the LKR domain, SDH domain, or both. Some monoclonal antibodies specifically target the LKR domain only .

• Application compatibility: Verify that the antibody has been validated for your specific application (Western blot, ELISA, immunohistochemistry, etc.) .

• Antibody type: Consider whether a monoclonal or polyclonal antibody is more suitable for your research. Monoclonal antibodies offer higher specificity but may be more sensitive to epitope modifications .

• Format: Determine if you need a conjugated (e.g., fluorophore, enzyme) or unconjugated antibody based on your detection method .

What are the optimal protocols for using LKR/SDH antibodies in Western blotting?

The optimal Western blotting protocol for LKR/SDH typically includes:

• Sample preparation: Extract proteins from tissues with high LKR/SDH expression (e.g., reproductive tissues or developing seeds in plants) . Use a buffer containing protease inhibitors to prevent degradation.

• Protein separation: Given the large size of LKR/SDH (102.1 kDa for human protein) , use a lower percentage SDS-PAGE gel (7-8%) for better resolution of high molecular weight proteins.

• Transfer: Employ a wet transfer method with longer transfer times (overnight at low voltage or 2 hours at higher voltage) to ensure complete transfer of the large protein.

• Blocking: Block with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody incubation: Dilute the LKR/SDH antibody as recommended (typically 1:1000 to 1:5000) and incubate overnight at 4°C.

• Detection: Use appropriate secondary antibodies and visualization methods based on your laboratory's capabilities.

• Controls: Include positive controls (tissue known to express LKR/SDH) and negative controls (LKR/SDH knockout tissue if available) .

How can researchers optimize immunohistochemistry protocols for LKR/SDH localization studies?

To optimize immunohistochemistry for LKR/SDH localization:

• Fixation: Use 4% paraformaldehyde for most tissues, but optimize fixation time based on tissue type (shorter for delicate tissues, longer for dense tissues).

• Antigen retrieval: Since LKR/SDH is mitochondrially localized , heat-induced epitope retrieval using citrate buffer (pH 6.0) is often necessary to expose mitochondrial antigens.

• Blocking: Block with serum from the same species as the secondary antibody (typically 5-10%) with 0.1-0.3% Triton X-100 for permeabilization.

• Primary antibody incubation: Incubate with LKR/SDH antibody at optimized dilution (typically 1:100 to 1:500) overnight at 4°C.

• Controls: Include mitochondrial markers for co-localization studies to confirm the expected subcellular localization pattern.

• Counterstaining: Use DAPI for nuclear staining and consider using MitoTracker or other mitochondrial dyes for co-localization confirmation.

• Visualization: Use confocal microscopy for precise subcellular localization, especially when confirming mitochondrial localization.

How can LKR/SDH antibodies be utilized to investigate tissue-specific expression patterns?

LKR/SDH antibodies can be employed in multiple complementary approaches to map tissue-specific expression:

• Immunohistochemistry in tissue sections: This allows visualization of LKR/SDH expression with cellular resolution across different tissue types. Research has shown that LKR/SDH is expressed in various tissues, with notable expression in appendix and duodenum in humans .

• Western blot analysis of tissue extracts: Quantitative comparison of LKR/SDH protein levels across different tissue types. This approach has been used to demonstrate differential expression levels between wild-type, heterozygous, and homozygous knockout plants .

• Flow cytometry of dissociated cells: For quantitative assessment of LKR/SDH expression in specific cell populations isolated from heterogeneous tissues.

• Immunoprecipitation followed by mass spectrometry: To identify tissue-specific post-translational modifications or interaction partners of LKR/SDH.

• Tissue microarray analysis: For high-throughput screening of LKR/SDH expression across multiple tissue samples simultaneously.

What strategies can researchers employ to study LKR/SDH protein-protein interactions?

To investigate LKR/SDH protein-protein interactions, researchers can implement:

• Co-immunoprecipitation (Co-IP): Use LKR/SDH antibodies to pull down the protein complex, followed by identification of interacting partners via Western blot or mass spectrometry.

• Proximity ligation assay (PLA): This technique can visualize and quantify protein interactions in situ with high sensitivity, useful for confirming interactions identified through other methods.

• Bimolecular fluorescence complementation (BiFC): Though not directly using antibodies, this approach can complement antibody-based methods to visualize protein interactions in living cells.

• Chromatin immunoprecipitation (ChIP): If investigating the reported transcriptional regulatory functions of LKR/SDH , ChIP with LKR/SDH antibodies can identify DNA binding sites.

• Cross-linking followed by immunoprecipitation: This approach captures transient interactions that might be missed by standard Co-IP protocols.

• Yeast two-hybrid validation: Confirm interactions identified through antibody-based methods using orthogonal approaches like Y2H.

How can researchers use LKR/SDH antibodies to investigate changes in protein expression under different physiological conditions?

To monitor LKR/SDH expression changes under varying physiological conditions:

• Quantitative Western blotting: Compare LKR/SDH protein levels across different conditions while normalizing to appropriate loading controls.

• ELISA assays: Develop quantitative ELISA protocols using LKR/SDH antibodies for high-throughput analysis of protein levels in multiple samples.

• Immunofluorescence intensity quantification: Measure changes in fluorescence intensity in tissue sections immunostained for LKR/SDH under different conditions.

• Flow cytometry: Quantify changes in LKR/SDH expression at the single-cell level using intracellular staining protocols.

• Proteomics approaches: Combine immunoprecipitation with mass spectrometry to identify condition-specific post-translational modifications.

• In vivo imaging: For animal models, develop protocols for non-invasive tracking of LKR/SDH expression using labeled antibodies.

This approach has been successfully employed to demonstrate that LKR/SDH gene expression is up-regulated in plant seeds and floral organs , providing insights into its role in reproduction and development.

What are common causes of false negative results in LKR/SDH immunodetection and how can they be addressed?

Several factors can contribute to false negative results when detecting LKR/SDH:

• Insufficient protein extraction: LKR/SDH is mitochondrially localized , requiring efficient organelle disruption. Solution: Use stronger lysis buffers containing detergents suitable for mitochondrial membrane solubilization.

• Epitope masking: Post-translational modifications or protein folding may obscure antibody binding sites. Solution: Test different antibodies targeting distinct epitopes or employ antigen retrieval methods.

• Low antibody sensitivity: The antibody may not be sensitive enough for your application. Solution: Try signal amplification methods such as tyramide signal amplification or polymer-based detection systems.

• Improper sample storage: Degradation of the target protein. Solution: Add protease inhibitors promptly during extraction and avoid repeated freeze-thaw cycles.

• Tissue-specific expression differences: LKR/SDH may not be expressed in your tissue of interest. Solution: Include positive control tissues with known LKR/SDH expression, such as reproductive tissues in plants .

• Antibody cross-reactivity assessment: Validate antibody specificity using tissues from knockout organisms where available .

How can researchers distinguish between LKR and SDH domains when studying this bifunctional protein?

To differentiate between the LKR and SDH domains of the bifunctional protein:

• Domain-specific antibodies: Use antibodies specifically raised against either the LKR or SDH domain. For example, monoclonal antibodies specifically recognizing the LKR domain have been successfully employed in research .

• Differential activity assays: Complement immunological detection with enzymatic activity assays specific for either LKR or SDH function.

• Recombinant expression of individual domains: Create positive controls by expressing the LKR or SDH domains separately for antibody validation.

• Peptide competition assays: Use synthetic peptides corresponding to specific domains to selectively block antibody binding and confirm specificity.

• Sequential immunoprecipitation: Use domain-specific antibodies in sequential immunoprecipitation to identify domain-specific interaction partners.

• Proteolytic fragmentation: Limited proteolysis followed by Western blotting with domain-specific antibodies can reveal differential stability or accessibility of the two domains.

What strategies can help resolve inconsistent results when comparing LKR/SDH antibody data with gene expression analysis?

When antibody-based protein detection doesn't align with gene expression data:

• Post-transcriptional regulation: Investigate microRNA regulation or RNA stability factors that might affect translation of LKR/SDH mRNA.

• Protein stability assessment: Examine protein half-life using pulse-chase experiments with LKR/SDH antibodies to detect the protein over time.

• Alternative splicing analysis: Design PCR primers to detect potential splice variants and corresponding antibodies to detect protein isoforms.

• Sensitivity differences: qPCR may detect low-level transcripts not resulting in detectable protein. Solution: Use more sensitive protein detection methods or enrichment approaches.

• Temporal disconnects: Consider time lags between transcription and translation. Solution: Perform time-course studies tracking both mRNA and protein levels.

• Technical validation: Cross-validate using multiple antibodies and gene expression assays to rule out technical artifacts.

Studies have shown that LKR/SDH knockout plants exhibited no detectable mRNA using Northern blot analysis and no detectable protein using Western blot analysis with specific antibodies, demonstrating concordance between transcript and protein levels in this case .

How can LKR/SDH antibodies contribute to improving nutritional quality in crop plants?

LKR/SDH antibodies are valuable tools for improving crop nutritional quality through:

• Screening for natural variants: Identify plants with altered LKR/SDH expression or activity that might accumulate higher lysine levels.

• Validating genetic modifications: Confirm reduced or abolished LKR/SDH protein levels in knockout or knockdown lines engineered for higher lysine content.

• Monitoring protein levels in breeding programs: Track LKR/SDH expression in crosses between high-lysine lines and elite cultivars.

• Tissue-specific expression analysis: Determine if LKR/SDH expression is reduced specifically in edible tissues while maintained in tissues important for plant vigor.

• Developmental expression profiling: Optimize harvest timing based on when LKR/SDH levels are naturally lower to maximize lysine content.

Research has demonstrated that knocking out the LKR/SDH gene in Arabidopsis resulted in significantly higher free and protein-incorporated lysine in seeds compared to wild-type plants, providing direct evidence for the significance of lysine catabolism in regulating lysine accumulation in plant seeds . This knowledge can be applied to crop improvement strategies aimed at enhancing nutritional quality.

What methodological approaches can researchers use to investigate the role of LKR/SDH in plant stress responses?

To study LKR/SDH's role in plant stress responses, researchers can implement:

• Stress-induced expression analysis: Use Western blotting with LKR/SDH antibodies to quantify protein levels in plants subjected to various stresses (drought, salt, pathogen infection).

• Subcellular localization under stress: Employ immunocytochemistry to track potential stress-induced changes in LKR/SDH localization.

• Post-translational modification analysis: Use phospho-specific or other PTM-specific antibodies alongside general LKR/SDH antibodies to detect stress-induced modifications.

• Comparative analysis between wild-type and LKR/SDH mutants: Expose both genotypes to stress conditions and compare physiological and molecular responses.

• Co-immunoprecipitation under stress conditions: Identify stress-specific interaction partners that might regulate LKR/SDH activity.

• Enzyme activity correlation: Combine immunoquantification with enzyme activity assays to determine if protein levels correlate with catalytic activity under stress.

How can researchers optimize experimental design when using LKR/SDH antibodies to study lysine metabolism in different plant tissues?

For optimal experimental design in plant tissue studies:

• Developmental timing: LKR/SDH expression is strongly up-regulated in floral organs and developing seeds . Sample collection should be timed accordingly to capture peak expression periods.

• Tissue selection: Focus on tissues with known LKR/SDH expression (reproductive tissues, developing seeds) but also include vegetative tissues as controls.

• Extraction buffer optimization: Different plant tissues require specific extraction protocols; optimize buffers for each tissue type while ensuring LKR/SDH stability.

• Comparative controls: Always include wild-type controls alongside any genetic variants being studied .

• Technical replication: Due to variability in plant samples, include sufficient biological and technical replicates (minimum n=3 for both).

• Quantification standards: Include recombinant LKR/SDH protein standards for absolute quantification when necessary.

• Validation with multiple methods: Complement Western blotting with immunohistochemistry or ELISA for more comprehensive analysis.

How might new antibody technologies enhance our understanding of LKR/SDH regulation and function?

Emerging antibody technologies offer new possibilities for LKR/SDH research:

• Single-domain antibodies (nanobodies): Their small size enables access to epitopes unavailable to conventional antibodies and facilitates intracellular tracking of LKR/SDH.

• Proximity-dependent labeling: Antibody-enzyme fusion proteins can be used to identify proteins in close proximity to LKR/SDH in its native cellular environment.

• Intrabodies: Antibodies engineered to function within living cells could be used to track or even modulate LKR/SDH activity in real-time.

• Degradation-targeting chimeric molecules: Antibody-based degraders could be developed for targeted degradation of LKR/SDH to study acute loss-of-function effects.

• Multiplex imaging technologies: New approaches allow simultaneous visualization of LKR/SDH alongside dozens of other proteins in the same sample.

• Antibody-guided CRISPR systems: Antibodies could direct gene editing machinery to the genomic regions near actively transcribing LKR/SDH genes.

What bioinformatic approaches can complement antibody-based studies of LKR/SDH structure and function?

Computational methods that enhance antibody-based LKR/SDH research include:

• Epitope prediction algorithms: Identify optimal antigenic regions for raising new, more specific antibodies against LKR/SDH domains.

• Structural modeling: Predict the three-dimensional structure of LKR/SDH to better understand epitope accessibility and domain interactions.

• Phylogenetic analysis: Compare LKR/SDH sequences across species to identify conserved regions as targets for antibodies with broad cross-reactivity.

• Network analysis: Integrate antibody-derived protein interaction data with transcriptomic and proteomic datasets to place LKR/SDH in broader biological networks.

• Machine learning approaches: Apply AI algorithms to immunohistochemistry images to quantify LKR/SDH expression patterns across different experimental conditions.

• Virtual screening: Identify small molecules that might modulate LKR/SDH activity by targeting specific domains identified through antibody studies.

How can researchers integrate LKR/SDH antibody data with multi-omics approaches for systems-level understanding?

Integration of antibody data with multi-omics for LKR/SDH research:

• Correlation analysis: Compare LKR/SDH protein levels detected by antibodies with transcriptome data to identify regulatory relationships.

• Proteogenomics: Combine genomic variation data with antibody-detected protein levels to identify genetic variants affecting LKR/SDH expression or stability.

• Metabolite correlation: Link LKR/SDH protein levels with metabolomics data focusing on lysine and its catabolic products to establish flux relationships.

• Tissue-specific integration: Create tissue-specific models incorporating antibody-derived localization data with tissue-specific transcriptome and proteome data.

• Perturbation studies: Use antibodies to quantify LKR/SDH in response to various perturbations, then integrate with corresponding transcriptomic and metabolomic changes.

• Temporal dynamics: Track LKR/SDH levels across developmental time points and integrate with developmental transcriptomics to identify key regulatory transitions.