SHM7 Antibody

What is SHM7 and why are antibodies against it important for plant research?

SHM7 (Serine hydroxymethyltransferase 7) is one of seven SHMT genes in Arabidopsis thaliana. This enzyme catalyzes the reversible conversion of serine and tetrahydrofolate (THF) to glycine and 5,10-methylene THF, playing a crucial role in one-carbon metabolism in plants . Antibodies against SHM7 are important tools for studying its expression, localization, and function in plant tissues. They allow researchers to:

• Detect native SHM7 protein in plant extracts

• Determine subcellular localization through immunohistochemistry

• Study protein-protein interactions involving SHM7

• Assess SHM7 expression under various environmental conditions or in different mutant backgrounds

The strategic importance of SHM7 in plant metabolism makes these antibodies valuable for understanding fundamental aspects of plant physiology and development.

How does SHM7 differ from other SHMT isoforms in plants?

SHM7 is one of seven SHMT isoforms (SHM1-SHM7) encoded in the Arabidopsis genome . These isoforms differ in:

SHM7 has unique sequence characteristics and potentially specialized functions compared to other isoforms. The antibodies against SHM7 are designed to recognize epitopes unique to this isoform, preventing cross-reactivity with other SHMT proteins .

What methods are used to generate and validate SHM7-specific antibodies?

SHM7 antibodies are typically generated through one of two approaches:

• Peptide Antibody Approach:

  • Synthetic peptides corresponding to unique regions of SHM7 are used as immunogens

  • Conjugated to carrier proteins (like KLH or BSA)

  • Injected into host animals (typically rabbits)

  • This approach has shown low success rates (~20-30%) for plant proteins

• Recombinant Protein Approach:

  • Partial or full-length SHM7 is expressed in E. coli expression systems

  • Proteins are purified using affinity tags

  • Used as immunogens in host animals

  • This approach shows higher success rates (50-60%) for plant proteins

Validation Methods:

• Western blot against plant tissue expressing SHM7

• Testing against SHM7 knockout/knockdown lines

• Dot blot assays against recombinant protein

• Cross-reactivity testing against other SHMT isoforms

• Immunolocalization studies compared with known expression patterns

The recombinant protein approach has generally shown better success for plant proteins. For SHM7 specifically, affinity purification of antibodies significantly improves detection sensitivity and specificity .

How can I determine if the SHM7 antibody is suitable for my specific experimental applications?

To assess the suitability of an SHM7 antibody for your research:

• Review validation data:

  • Check if the antibody has been validated for your application (Western blot, immunoprecipitation, immunolocalization)

  • Look for positive controls using known SHM7-expressing tissues

  • Check for negative controls using SHM7 knockout/knockdown lines

• Perform preliminary validation:

  • Run a Western blot using tissues known to express SHM7

  • Include appropriate positive and negative controls

  • Verify band size corresponds to predicted SHM7 molecular weight

  • Test detection limits using serial dilutions

• Application-specific testing:

  • For immunolocalization: Test fixation conditions and antibody concentrations

  • For co-immunoprecipitation: Verify pull-down efficiency with known interactors

  • For ELISA: Establish standard curves with recombinant protein

• Cross-reactivity assessment:

  • Test against tissues expressing other SHMT isoforms

  • Consider using tissues from other plant species if cross-species reactivity is desired

This systematic approach ensures the antibody will perform reliably in your specific experimental system before proceeding with full-scale experiments .

What are the optimal conditions for using SHM7 antibodies in Western blot analyses of plant tissues?

For optimal Western blot results with SHM7 antibodies:

Sample Preparation:

• Extract proteins in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, with protease inhibitors

• For Arabidopsis, use young seedlings or specific tissues with known SHM7 expression

• Include phosphatase inhibitors if phosphorylation status is important

Gel Electrophoresis and Transfer:

• Use 10-12% SDS-PAGE gels for optimal resolution

• Transfer to PVDF membranes (better protein retention compared to nitrocellulose)

• Transfer at 100V for 1-2 hours or 30V overnight at 4°C

Blocking and Antibody Incubation:

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

• Dilute primary SHM7 antibody 1:1000 to 1:5000 in blocking solution

• Incubate overnight at 4°C with gentle agitation

• Wash 4-5 times with TBST, 5 minutes each

• Incubate with HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

Detection and Troubleshooting:

• Use ECL detection systems (standard or high sensitivity depending on expression level)

• Expected molecular weight for SHM7 is approximately 50-55 kDa

• Multiple bands may indicate post-translational modifications or degradation

• Absence of signal may require longer exposure or higher antibody concentration

• High background may require more stringent washing or lower antibody concentration

Always include appropriate controls, such as recombinant SHM7 protein as a positive control and extracts from SHM7 knockout plants as negative controls .

How can SHM7 antibodies be effectively used for immunolocalization studies in plant tissues?

For successful immunolocalization of SHM7 in plant tissues:

Tissue Preparation:

• Fix tissues in 4% paraformaldehyde in PBS for 1-2 hours

• For Arabidopsis roots: fix for 30-45 minutes to preserve structural integrity

• Embed in paraffin or prepare for cryo-sectioning

• Section to 5-10 μm thickness for optimal antibody penetration

Antigen Retrieval and Blocking:

• Perform antigen retrieval if needed: 10 mM sodium citrate buffer (pH 6.0) for 10-15 minutes

• Block with 2-5% BSA in PBS with 0.1% Triton X-100 for 1 hour

• For high background: include 5-10% normal serum from the species of secondary antibody

Antibody Incubation:

• Dilute affinity-purified SHM7 antibody 1:50 to 1:200 in blocking solution

• Incubate overnight at 4°C in a humid chamber

• Wash 3-4 times with PBS containing 0.1% Triton X-100

• Apply fluorophore-conjugated secondary antibody (1:200-1:500) for 1-2 hours at room temperature

• Include DAPI (1 μg/ml) for nuclear counterstaining

Controls and Visualization:

• Include sections from SHM7 knockout plants as negative controls

• Process control slides without primary antibody to assess background

• Use confocal microscopy for co-localization studies with organelle markers

• Expected pattern: punctate plastid localization in specific cell types

Technical Considerations:

• Affinity-purified antibodies significantly improve signal-to-noise ratio

• Signal amplification methods (e.g., tyramide signal amplification) may be needed for low-abundance SHM7

• Cross-linking fixatives may mask epitopes; optimize fixation times

• For whole-mount immunostaining of Arabidopsis roots, permeabilization steps may need optimization

This approach has been successfully used for localizing various plant proteins with similar characteristics to SHM7 .

How can SHM7 antibodies be used in chromatin immunoprecipitation (ChIP) experiments for studying protein-DNA interactions?

While SHM7 itself is not known to be a DNA-binding protein, this methodology applies to studying proteins that may interact with SHM7 in chromatin contexts:

Protocol Optimization:

• Crosslink plant tissue with 1% formaldehyde for 10-15 minutes

• Quench with 0.125 M glycine for 5 minutes

• Isolate nuclei using sucrose gradient centrifugation

• Sonicate chromatin to 200-500 bp fragments

• Pre-clear with protein A/G beads

• Immunoprecipitate with SHM7 antibody (5-10 μg per reaction)

• Include negative controls: IgG from same species, no-antibody control

Critical Considerations:

• Validate antibody specificity for ChIP applications specifically

• Test different crosslinking times and sonication conditions

• Verify protein-DNA interactions using sequential ChIP or reverse ChIP

• Include input controls and normalization genes

Data Analysis and Interpretation:

• Analyze enrichment by qPCR or next-generation sequencing

• Use appropriate statistical methods for peak calling

• Consider biological replicates to establish reproducibility

• Validate findings with orthogonal approaches (e.g., EMSA, reporter assays)

This approach has been successfully used for studying various plant proteins, including those involved in metabolic regulation and stress responses .

What approaches can be used to analyze potential post-translational modifications of SHM7 using specific antibodies?

To study post-translational modifications (PTMs) of SHM7:

Antibody-Based Approaches:

• Generate modification-specific antibodies (e.g., phospho-SHM7, acetyl-SHM7)

• Use PTM-specific antibodies in combination with SHM7 antibodies

• Perform immunoprecipitation with SHM7 antibody followed by Western blot with PTM-specific antibodies

Sample Preparation:

• Include phosphatase inhibitors (50 mM NaF, 1 mM Na₃VO₄) for phosphorylation studies

• Add deacetylase inhibitors (5-10 mM sodium butyrate, 1 μM TSA) for acetylation studies

• Add proteasome inhibitors (10-50 μM MG132) for ubiquitination studies

• Use fresh tissue and maintain samples at 4°C

Analytical Techniques:

• 2D gel electrophoresis followed by Western blotting

• Phos-tag SDS-PAGE for detecting phosphorylated forms

• Immunoprecipitation coupled with mass spectrometry

• Sequential immunoprecipitation with different modification-specific antibodies

Data Interpretation:

• Compare PTM patterns under different conditions (development, stress, etc.)

• Correlate modifications with enzyme activity measurements

• Validate using site-directed mutagenesis of modified residues

• Compare with known PTM sites of other SHMT isoforms

This approach has been successfully applied to study PTMs in various plant enzymes involved in primary metabolism .

What strategies can resolve non-specific binding or weak signal issues when using SHM7 antibodies?

For Non-specific Binding Issues:

For Weak Signal Issues:

General Improvement Strategies:

• Affinity purify antibodies against the recombinant protein

• Use fresh tissue samples for optimal protein preservation

• Optimize protein extraction buffers for SHM7 solubilization

• Consider using signal amplification methods (e.g., TSA, enhanced chemiluminescence)

• Include appropriate positive and negative controls

Research has shown that affinity purification significantly improves detection rate and specificity for plant protein antibodies, with success rates improving from below 30% to over 50% .

How can I validate antibody specificity for SHM7 when knockout/mutant lines are unavailable?

When SHM7 knockout/mutant lines are unavailable, consider these alternative validation approaches:

Gene Silencing Approaches:

• Use RNA interference (RNAi) to knockdown SHM7 expression

• Employ virus-induced gene silencing (VIGS) for temporary knockdown

• Utilize CRISPR/Cas9 to generate transient knockouts

• Compare antibody signal between silenced and wild-type plants

Competition Assays:

• Pre-incubate antibody with excess recombinant SHM7 protein

• Observe elimination of specific signal on Western blot or immunostaining

• Include unrelated proteins as negative controls in competition

Heterologous Expression:

• Express SHM7 in heterologous systems (E. coli, yeast, etc.)

• Compare signal between expressing and non-expressing cells

• Include epitope tags for parallel detection with tag-specific antibodies

Correlation with Transcript Levels:

• Compare antibody signal in tissues with known differential SHM7 expression

• Correlate protein detection with RT-qPCR measurements

• Use conditions known to induce/repress SHM7 expression

Cross-Reactivity Testing:

• Test antibody against recombinant proteins of all SHMT isoforms

• Examine tissues with differential expression of various SHMT isoforms

• Perform peptide array analysis to confirm epitope specificity

These approaches, particularly when used in combination, can provide strong evidence for antibody specificity even without knockout lines .

How can quantitative analysis of SHM7 expression be performed across different tissues or environmental conditions?

For quantitative analysis of SHM7 expression:

Western Blot Quantification:

• Use loading controls (ACTIN, TUBULIN, or GAPDH)

• Include recombinant SHM7 protein standards for absolute quantification

• Employ fluorescent secondary antibodies for wider linear range

• Analyze with appropriate software (ImageJ, Image Studio Lite)

• Normalize signal to total protein using stain-free gels or Ponceau staining

ELISA-Based Quantification:

• Develop sandwich ELISA using two different SHM7 antibodies

• Create standard curves with recombinant SHM7 protein

• Analyze multiple biological and technical replicates

• Account for matrix effects in different tissue extracts

Immunohistochemistry Quantification:

• Use consistent image acquisition parameters

• Quantify signal intensity relative to background

• Employ automated image analysis software

• Consider cell-type specific quantification

Experimental Design Considerations:

• Include time-course analyses for dynamic responses

• Compare multiple tissues or cell types simultaneously

• Control for developmental stage and environmental conditions

• Include appropriate statistical analyses (ANOVA, mixed models)

Data Presentation:

• Present absolute concentration when possible

• Show relative changes with appropriate error bars

• Include representative images alongside quantification

• Correlate protein levels with enzymatic activity assays

This quantitative approach allows precise measurement of SHM7 expression changes in response to developmental or environmental cues .

What are the considerations for studying protein-protein interactions involving SHM7 using co-immunoprecipitation approaches?

For studying SHM7 protein-protein interactions:

Sample Preparation:

• Use mild lysis buffers to preserve protein interactions (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% NP-40)

• Include protease and phosphatase inhibitors

• Minimize time between extraction and immunoprecipitation

• Consider crosslinking approaches (formaldehyde, DSP) for transient interactions

Immunoprecipitation Protocol:

• Pre-clear lysates with protein A/G beads

• Use 2-5 μg SHM7 antibody per 200-500 μg protein extract

• Incubate overnight at 4°C with gentle rotation

• Wash beads carefully to remove non-specific interactions

• Elute with gentle conditions to maintain interacting proteins

Controls and Validation:

• Include negative controls: IgG from same species, unrelated antibody

• Use SHM7 knockout/knockdown extracts as specificity controls

• Perform reciprocal co-immunoprecipitation with antibodies against putative interactors

• Validate interactions with orthogonal methods (Y2H, BiFC, FRET)

Analysis of Interacting Partners:

• Identify by mass spectrometry or specific antibodies

• Classify interactions based on biological function

• Map interaction domains through deletion constructs

• Assess interaction dynamics under different conditions

Methodological Variations:

• Tandem affinity purification for higher stringency

• Proximity-dependent biotin labeling (BioID) for weak/transient interactions

• Size exclusion chromatography coupled with immunoprecipitation

• Native vs. denaturing conditions for different interaction types

This approach can reveal the protein interaction network centered around SHM7, providing insights into its functional regulation and integration within metabolic pathways .

How can SHM7 antibodies be used for evolutionary studies across different plant species?

For evolutionary studies using SHM7 antibodies:

Cross-Species Reactivity Assessment:

• Test antibody against protein extracts from diverse plant species

• Verify expected molecular weight shifts based on sequence differences

• Consider using antibodies against conserved regions for wider cross-reactivity

• Generate phylogenetic trees based on epitope conservation

Comparative Expression Analysis:

• Analyze SHM7 expression patterns across related species

• Compare subcellular localization in different plant lineages

• Assess post-translational modifications across evolutionary distance

• Correlate expression with metabolic adaptations

Technical Adaptations:

• Optimize extraction buffers for different plant tissues

• Adjust antibody concentrations based on cross-reactivity efficiency

• Consider using secondary antibodies designed for multi-species studies

• Validate specificity in each species using available genetic resources

Data Interpretation Framework:

• Map protein expression differences onto established phylogenies

• Correlate structural conservation with functional conservation

• Analyze convergent/divergent expression patterns across lineages

• Consider timing of gene duplication events in data interpretation

This approach enables tracking evolutionary changes in SHM7 expression, localization, and function across plant lineages, providing insights into adaptive metabolic evolution .

What considerations are important when comparing data from antibody-based detection with transcript-level analyses of SHM7?

When comparing protein-level (antibody-based) and transcript-level data:

Biological Considerations:

• Protein and mRNA have different half-lives (typically hours vs. minutes)

• Post-transcriptional regulation may affect protein abundance

• Translational efficiency varies under different conditions

• Post-translational modifications affect antibody detection but not transcripts

Technical Considerations:

• Antibody detection has different sensitivity than RT-qPCR

• Dynamic range differs between protein and transcript detection methods

• Cell-type specificity may be more easily achieved with transcriptomics

• Normalization methods differ between proteomics and transcriptomics

Experimental Design for Integrated Analysis:

• Collect samples for both analyses simultaneously

• Include time-course studies to capture delayed protein response

• Analyze multiple biological replicates to establish correlations

• Consider cell-type specific approaches when possible

Data Integration Framework:

• Calculate Spearman/Pearson correlations between protein and transcript

• Classify genes as concordant or discordant in regulation

• Apply time-delay analyses for dynamic responses

• Consider mathematical modeling to capture regulatory relationships

Interpretation Guidelines:

• Discordance suggests post-transcriptional regulation

• Strong correlation indicates primarily transcriptional control

• Temporal shifts suggest ordered regulation

• Tissue-specific differences may reflect differential regulation

This integrated approach provides a more comprehensive understanding of SHM7 regulation at multiple levels, revealing both transcriptional and post-transcriptional control mechanisms .

How might new antibody technologies enhance SHM7 research in plant systems?

Emerging antibody technologies offer exciting possibilities for SHM7 research:

Nanobodies and Single-Domain Antibodies:

• Smaller size allows better tissue penetration

• Higher stability under varied experimental conditions

• Enhanced access to cryptic epitopes

• Potential for direct fusion to fluorescent proteins for live imaging

Engineered Recombinant Antibodies:

• Rational design for enhanced specificity to SHM7

• Humanized constant regions for reduced background in plant tissues

• Affinity maturation for improved sensitivity

• Bi-specific antibodies for simultaneous detection of SHM7 and interacting partners

Intrabodies for In Vivo Applications:

• Expression of antibody fragments within living plant cells

• Targeting to specific subcellular compartments

• Potential for protein function modulation

• Real-time monitoring of protein dynamics

Advanced Conjugation Technologies:

• Site-specific conjugation for consistent orientation

• Novel fluorophores with improved quantum yield and photostability

• Bifunctional labels for correlative microscopy

• Enzyme-coupled antibodies for amplified detection

Application-Specific Adaptations:

• Super-resolution microscopy compatible antibodies

• Mass cytometry compatible metal-conjugated antibodies

• Electron microscopy optimized formulations

• Quantitative imaging standards for absolute quantification

These technologies have the potential to greatly enhance the specificity, sensitivity, and applications of SHM7 antibodies in plant research .

What methodological approaches allow integration of antibody-based SHM7 detection with multi-omics studies?

To integrate antibody-based SHM7 detection with multi-omics:

Sample Preparation Strategies:

• Develop unified extraction protocols compatible with multiple analyses

• Consider sequential extraction from the same tissue

• Use microsampling techniques for spatial correlation

• Implement single-cell approaches when feasible

Integrated Experimental Design:

• Coordinate sampling timepoints across all platforms

• Include common reference samples for cross-platform normalization

• Design perturbations that affect multiple molecular levels

• Consider both acute and chronic responses

Technical Integration Approaches:

• Spatial transcriptomics with immunofluorescence

• Immuno-CITE-seq for combined protein and RNA profiling

• Antibody-based sorting followed by omics analysis

• Activity-based protein profiling with antibody validation

Data Analysis Frameworks:

• Develop unified data models across molecular levels

• Apply multi-block statistical methods

• Utilize machine learning for integrative pattern discovery

• Implement network-based integration approaches

Biological Interpretation Strategies:

• Map protein-level data onto pathway models

• Correlate protein abundance with metabolic flux changes

• Integrate with chromatin structure and accessibility data

• Connect to phenotypic outcomes at cellular and organismal levels

This integrated approach provides a systems-level understanding of SHM7 function in plant metabolism and development, revealing regulatory connections across multiple molecular levels .