SHMT1 Antibody

What is SHMT1 and why is it important in metabolic research?

SHMT1 (cytoplasmic serine hydroxymethyltransferase) is an enzyme that catalyzes the interconversion of serine and glycine while transferring a one-carbon unit to tetrahydrofolate . This reaction is fundamental to one-carbon metabolism and plays critical roles in:

• Regulating the partitioning of folate-activated one-carbons between thymidylate and S-adenosylmethionine biosynthesis

• Maintaining cellular methylation potential and genome stability

• Supporting nucleotide synthesis necessary for DNA replication and repair

• Contributing to cancer metabolic reprogramming through amino acid metabolism

• Supporting neurogenesis and cognitive function

SHMT1 expression patterns and activity levels directly affect these processes, making it a significant target in metabolic, cancer, and neuroscience research.

What are the validated applications for SHMT1 antibodies?

Based on current research literature, SHMT1 antibodies have been validated for the following applications:

These applications enable researchers to detect, quantify, and localize SHMT1 protein in various experimental contexts.

How should I select an appropriate SHMT1 antibody for my research?

When selecting an SHMT1 antibody, consider these critical factors:

• Target epitope: Different antibodies target specific regions of SHMT1. For example, ab186130 targets the C-terminal region (aa 450 to C-terminus) , while ab224445 targets the N-terminal region (aa 1-100) . This distinction is important if you're investigating potential cleavage products, fusion proteins, or specific domains.

• Species reactivity: Ensure the antibody is validated for your species of interest. Currently available antibodies have been validated for human and mouse samples , but cross-reactivity with other species may vary.

• Application compatibility: Verify that the antibody has been validated for your specific application. Not all antibodies perform equally across different techniques.

• Validation data: Review the quality of validation data, including predicted band size (53 kDa for SHMT1) , positive controls (e.g., Jurkat cells), and published citations.

• Host species: Consider the host species (typically rabbit for currently available polyclonal antibodies) to avoid cross-reactivity issues in multi-labeling experiments.

What controls are essential for validating SHMT1 antibody specificity?

To ensure the reliability of your SHMT1 antibody experiments, incorporate these essential controls:

• Positive control: Use samples known to express SHMT1, such as Jurkat whole cell lysate (50 μg) .

• Negative control: Include samples from SHMT1 knockout/knockdown models when available, or use non-immune serum from the same species as the primary antibody .

• Loading control: For Western blotting, include a housekeeping protein such as GAPDH for normalization .

• Isotype control: For immunoprecipitation, include a control reaction with non-specific IgG from the same species as the SHMT1 antibody .

• Secondary antibody control: Perform a control omitting the primary antibody to assess non-specific binding of the secondary antibody.

These controls enable proper interpretation of results and help troubleshoot any technical issues.

How can SHMT1 antibodies be used to investigate metabolic reprogramming in cancer?

SHMT1 plays a central role in cancer metabolic reprogramming through its regulation of one-carbon metabolism. Researchers can leverage SHMT1 antibodies to investigate this process through several advanced approaches:

• Subcellular compartmentalization studies: SHMT1 can function in both the cytoplasm and nucleus , with compartmentalization regulated by SUMO-mediation during S-phase . Use immunofluorescence with SHMT1 antibodies to track this dynamic localization in cancer cells versus normal cells.

• Metabolic complex formation: SHMT1 participates in multi-enzyme complexes that regulate metabolic flux. Use co-immunoprecipitation with SHMT1 antibodies to identify protein interaction partners that may be altered in cancer cells.

• RNA-protein interaction analysis: The recently discovered interaction between SHMT1 protein and RNA molecules, particularly SHMT2 mRNA , represents a novel regulatory mechanism. Researchers can use SHMT1 antibodies in RNA immunoprecipitation (RIP) assays to characterize the cancer-specific RNA interactome of SHMT1.

• Tumor microenvironment effects: Correlate SHMT1 expression patterns with metabolic markers and tumor progression using IHC in tissue microarrays, which may reveal tissue-specific roles in different cancer types.

These approaches can provide mechanistic insights into how cancer cells exploit SHMT1 to "fine tune amino acids availability according to their metabolic needs" .

What methodological considerations are important when investigating SHMT1-RNA interactions?

The discovery that SHMT1 protein can interact with RNA molecules, particularly SHMT2 mRNA , reveals a complex layer of metabolic regulation. Researchers investigating this phenomenon should consider:

• RNA binding competition: SHMT1 can bind multiple RNA molecules with varying affinities following a Gaussian distribution . Design experiments that account for the presence of competing RNA species in the cellular environment.

• Quantitative binding analysis: Employ quantitative methods like surface plasmon resonance (SPR) or microscale thermophoresis (MST) to determine binding constants for SHMT1-RNA interactions, which can help predict regulatory outcomes.

• Metabolic consequences: Monitor how changes in RNA binding affect SHMT1 enzymatic activity and metabolite levels, particularly serine and glycine concentrations . This requires combining RNA-protein interaction studies with metabolomic analysis.

• Computational modeling: Integrate experimental data with stochastic dynamic models (e.g., Gillespie algorithm) to predict how RNA binding dynamically regulates SHMT1 activity in different cellular contexts.

• Validation strategies: Confirm computational predictions in relevant biological models, such as the lung adenocarcinoma cell line H1299 , using SHMT1 antibodies to track protein dynamics and interactions.

How can SHMT1 antibodies help elucidate the enzyme's role in neurogenesis and cognitive function?

SHMT1 has been implicated in hippocampal neurogenesis and cognitive function . Researchers can use SHMT1 antibodies to investigate this connection through several approaches:

• Developmental expression profiling: Use immunohistochemistry with SHMT1 antibodies on brain sections from different developmental stages to map temporal expression patterns, complementing in situ hybridization data for SHMT1 mRNA .

• Cellular specificity analysis: Combine SHMT1 antibody staining with neuronal, glial, and progenitor cell markers to determine which cell types express SHMT1 during neurogenesis.

• Activity-dependent regulation: Examine whether SHMT1 expression or localization changes in response to neuronal activity or learning paradigms using immunofluorescence in appropriate behavioral models.

• Signaling pathway interactions: Use co-immunoprecipitation with SHMT1 antibodies to identify neuronal-specific protein interactions that might explain its role in brain development and function.

• Comparative analysis in SHMT1-deficient models: Compare protein expression of neurogenesis markers and metabolic enzymes in wild-type versus Shmt1-deficient mice to identify compensatory mechanisms or downstream effectors.

What experimental approaches can resolve contradictory findings about SHMT1 reaction directionality?

Research indicates that "in some cell types the SHMT1 reaction is reversible, while in others it is only directed towards serine synthesis" . This contradiction can be addressed through several methodological approaches:

• Metabolic flux analysis: Use isotope-labeled precursors (13C-serine or 13C-glycine) to track directional flux through SHMT1 in different cell types, combined with immunoprecipitation to isolate SHMT1-associated metabolites.

• RNA-binding profiling: Quantify cell type-specific differences in SHMT1-RNA interactions using SHMT1 antibodies in RIP-seq experiments, correlating binding patterns with reaction directionality.

• Post-translational modification analysis: Immunoprecipitate SHMT1 from different cell types and analyze post-translational modifications by mass spectrometry to identify modifications that might affect enzyme directionality.

• Microenvironmental manipulation: Systematically vary metabolite concentrations, pH, and redox conditions while monitoring SHMT1 activity direction to identify environmental factors that influence reaction preference.

• Structure-function analysis: Generate site-directed mutants of SHMT1 to test hypotheses about structural determinants of directionality, using antibodies that recognize specific domains to track conformational changes.

What are the optimal conditions for Western blotting with SHMT1 antibodies?

For successful Western blot detection of SHMT1, follow these optimized protocol parameters:

For optimal results, always prepare fresh lysates, maintain consistent protein loading, and include appropriate positive and negative controls.

What protocols yield optimal results for immunohistochemistry with SHMT1 antibodies?

For high-quality immunohistochemical detection of SHMT1 in tissue sections:

• Tissue preparation:

  • Fix tissues in 10% neutral-buffered formalin

  • For intestinal tissues, the "Swiss roll" technique provides comprehensive visualization

  • Embed in paraffin and section at 5 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is typically effective

  • Optimize time and temperature based on tissue type

• Blocking and antibody incubation:

  • Block with appropriate serum (e.g., 5% normal goat serum)

  • Use ab224445 at 1:200 dilution or optimize concentration with titration

  • Incubate overnight at 4°C in a humidified chamber

• Detection and visualization:

  • For chromogenic detection, AEC (3-amino-9-ethylcarbazole) developer works well

  • Counterstain with Gill's hematoxylin #2

  • Mount with Fluoromount-G

• Controls:

  • Include non-immune serum from the same species as the primary antibody as a negative control

  • Include known positive tissue samples (human testis or kidney show good SHMT1 expression)

How can immunoprecipitation with SHMT1 antibodies be optimized for protein interaction studies?

For effective immunoprecipitation of SHMT1 and its interaction partners:

• Lysate preparation:

  • Use 1 mg of whole cell lysate (e.g., Jurkat cells)

  • Prepare lysate in a buffer that preserves protein-protein interactions (containing protease inhibitors and mild detergents)

• Pre-clearing step:

  • Pre-clear lysate with Protein A/G beads to reduce non-specific binding

  • Remove beads by centrifugation before adding SHMT1 antibody

• Antibody incubation:

  • Add 6 μg of SHMT1 antibody (e.g., ab186130) per reaction

  • Incubate with rotation overnight at 4°C to maximize binding

• Bead capture and washing:

  • Add pre-equilibrated Protein A/G beads and incubate with rotation

  • Perform 4-5 stringent washes to remove non-specific proteins

  • Include a final wash with buffer containing no detergent

• Elution and analysis:

  • Elute bound proteins with SDS sample buffer

  • Load approximately 20% of immunoprecipitated material for Western blot detection

  • Probe with SHMT1 antibody at 1 μg/ml for detection

• Controls:

  • Include a control using non-specific IgG from the same species as the SHMT1 antibody

  • Consider using SHMT1-deficient samples as negative controls when available

What experimental design considerations are important when using SHMT1-deficient models?

When working with SHMT1 knockout or deficient models, implement these methodological approaches for robust results:

• Model selection:

  • Complete knockout (Shmt1−/−) models show more severe phenotypes than hemizygous (Shmt1+/−) models

  • Select the appropriate model based on your specific research question and viability concerns

• Experimental groups:

  • Include wild-type littermates as controls

  • Consider including hemizygous animals as an intermediate phenotype group

  • Use age-matched and sex-matched animals (6-8 weeks of age is common for adult studies)

• Phenotypic assessment:

  • For inflammatory models: monitor weight loss, tissue damage, and infiltrated immune cells

  • For neurogenesis studies: assess cognitive function and hippocampal development

  • For metabolic studies: analyze serine/glycine levels and AdoMet concentrations

• Molecular analysis:

  • Assess changes in related enzymes (thymidylate synthase, cytoplasmic thymidine kinase)

  • Evaluate compensatory mechanisms that may mask phenotypes

  • Use multiple techniques (qPCR, Western blotting, metabolomics) for comprehensive analysis

• Statistical considerations:

  • Include sufficient biological replicates (minimum 3 animals per group)

  • Use appropriate statistical tests based on data distribution

  • Consider power analysis to determine sample size requirements

How can researchers troubleshoot common issues with SHMT1 antibody experiments?

When troubleshooting, systematically modify one variable at a time and maintain detailed records of all protocol modifications.

How should researchers interpret discrepancies between SHMT1 mRNA and protein levels?

Discrepancies between SHMT1 mRNA (measured by qPCR or in situ hybridization ) and protein levels (detected by SHMT1 antibodies) may reflect important biological mechanisms:

• Post-transcriptional regulation:

  • SHMT1 protein interacts with various RNA molecules, including SHMT2 mRNA

  • This RNA binding may affect translation efficiency of multiple transcripts in the cell

  • Investigate RNA binding factors that might regulate SHMT1 mRNA translation

• Protein stability and turnover:

  • SHMT1 protein half-life may vary across cell types or conditions

  • Perform cycloheximide chase experiments to assess protein stability

  • Investigate potential ubiquitination or other degradation signals

• Subcellular relocalization:

  • SHMT1 can shuttle between cytoplasm and nucleus, particularly during S-phase

  • Use fractionation methods combined with Western blotting to assess distribution

  • Apparent changes in protein levels may reflect redistribution rather than synthesis/degradation

• Methodological considerations:

  • Validate mRNA measurements with multiple primer sets

  • Use antibodies targeting different SHMT1 epitopes to confirm protein findings

  • Consider absolute quantification methods for both mRNA and protein

What statistical approaches are appropriate for analyzing SHMT1 expression data?

For robust statistical analysis of SHMT1 expression data:

• For comparative studies:

  • Use unpaired t-tests for comparing two groups with normal distribution

  • Apply one-way ANOVA with appropriate post-hoc tests for multiple group comparisons

  • Consider non-parametric alternatives (Mann-Whitney or Kruskal-Wallis) if data is not normally distributed

• For correlation analysis:

  • Use Pearson's correlation for normally distributed data

  • Apply Spearman's rank correlation for non-parametric data

  • Visualize correlations with scatter plots including regression lines

• For longitudinal studies:

  • Implement repeated measures ANOVA or mixed models

  • Account for time-dependent changes and subject variability

• Sample size considerations:

  • Include at least 3 biological replicates per group

  • Perform power analysis to determine adequate sample size

  • Report precise p-values rather than significance thresholds

• Data presentation:

  • Present results as mean ± SEM for normally distributed data

  • Use GraphPad Prism or similar software for statistical analysis and visualization

  • Consider indicating individual data points alongside group means

How does the discovery of SHMT1's RNA binding capacity change experimental approaches?

The finding that SHMT1 protein can bind RNA molecules introduces new dimensions to SHMT1 research that require innovative experimental approaches:

• Integrated omics approaches:

  • Combine RIP-seq (to identify SHMT1-bound RNAs) with metabolomics (to assess metabolic consequences)

  • Integrate proteomic and transcriptomic data to identify coordinated regulatory networks

• Structural biology considerations:

  • Investigate the structural basis of SHMT1-RNA interactions using crystallography or cryo-EM

  • Design structure-based mutations to selectively disrupt RNA binding without affecting enzymatic activity

• Dynamic regulatory studies:

  • Develop live-cell imaging approaches to visualize SHMT1-RNA interactions in real-time

  • Use optogenetic tools to modulate SHMT1-RNA binding and observe metabolic consequences

• Computational modeling:

  • Expand stochastic modeling approaches to predict system behavior under different conditions

  • Integrate models with experimental data to generate testable hypotheses about metabolic regulation

• Therapeutic implications:

  • Explore the potential of targeting SHMT1-RNA interactions in diseases with metabolic dysregulation

  • Screen for small molecules that modulate these interactions as potential therapeutic leads

This discovery fundamentally shifts our understanding of SHMT1 from a pure metabolic enzyme to a multifunctional protein with RNA-binding regulatory capabilities.

What are the emerging connections between SHMT1 and immune function?

Recent research has begun to reveal important roles for SHMT1 in immune function:

• B cell-mediated responses:

  • SHMT1-deficient B cells have been associated with more severe inflammatory damage

  • This suggests SHMT1 may have protective functions in B cell-mediated immune responses

• Macrophage infiltration and function:

  • SHMT1 deficiency affects macrophage infiltration in inflammatory contexts

  • Further investigation is needed to determine if this reflects direct regulation or indirect metabolic effects

• Oxidative stress connections:

  • Oxidative stress appears linked to SHMT1-mediated one-carbon metabolism in immune contexts

  • This connection may explain how metabolic alterations affect inflammatory responses

• Metabolic regulation of immune cell function:

  • One-carbon metabolism supports immune cell proliferation and effector functions

  • SHMT1's role in regulating serine/glycine availability may be critical for immune cell activation and differentiation

• Potential therapeutic applications:

  • Modulating SHMT1 activity could represent a novel approach to managing inflammatory conditions

  • Metabolic interventions targeting one-carbon metabolism might complement existing immunomodulatory strategies