DIN11 Antibody

What is DIN11 and why are antibodies against it valuable for plant research?

DIN11 is a plant homoarginine-6-hydroxylase that catalyzes the production of guanidine and glutamate semialdehyde (GSA). It belongs to the 2-ODD-C23 family of enzymes . DIN11 expression is induced by various stress conditions including prolonged darkness, wounding, and reactive oxygen species (ROS) accumulation .

Antibodies against DIN11 are valuable research tools because they enable:

• Tracking DIN11 protein expression under various stress conditions

• Determining subcellular localization through immunohistochemistry

• Distinguishing between DIN11 isoforms (Din11L and Din11s)

• Studying protein-protein interactions involving DIN11

• Validating gene knockout or silencing experiments

The ability to detect DIN11 protein levels provides critical data that complements transcriptomic analyses, offering insights into post-transcriptional regulation mechanisms in plant stress responses.

How can researchers generate specific antibodies against DIN11?

Generating specific antibodies against DIN11 requires careful antigen design, particularly due to the existence of different DIN11 isoforms and homologous proteins:

Recommended protocol:

• Antigen design: Analyze DIN11 sequence to identify unique epitopes not shared with homologs (At3g49630 and At3g50210) . The N-terminal region shows variability between Din11L and Din11s isoforms, making it suitable for isoform-specific antibodies .

• Peptide synthesis: Generate ~15-mer peptides representing unique regions of DIN11. For isoform-specific antibodies, utilize the first 24 amino acids unique to Din11L or the region around the second ATG start codon for Din11s .

• Immunization: Follow a protocol similar to that described in search result , involving multiple immunizations of rabbits with the synthesized peptides.

• Antibody purification: Perform affinity purification using the immunizing peptides to ensure specificity.

• Validation: Test antibody specificity against recombinant Din11L and Din11s proteins expressed in E. coli, as well as against protein extracts from wild-type and din11 mutant plants .

What are the optimal samples for DIN11 antibody validation?

Rigorous validation of DIN11 antibodies requires multiple complementary approaches:

• Recombinant protein samples:

  • Purified His-tagged DIN11 expressed in E. coli SoluBL21 cells

  • Both Din11L and Din11s isoforms for specificity testing

  • Related 2-ODD-C23 family members (At3g49630, At3g50210) as negative controls

• Plant tissue samples:

  • Arabidopsis wild-type plants (Columbia-0 ecotype) as positive control

  • CRISPR/Cas9-generated din11 knockout mutants created using pHEE401E vector system as negative controls

  • Plants subjected to dark treatment, wounding, or ROS-inducing conditions to test for increased DIN11 detection

• Subcellular fractions:

  • Cytosolic, nuclear, and membrane fractions to determine localization

  • Tissue microarrays (TMAs) as used for other antibody validations

For optimal results, collect Arabidopsis samples at multiple time points following stress treatments, as DIN11 expression increases during stress responses.

How can researchers distinguish between Din11L and Din11s isoforms in experimental systems?

Distinguishing between the two DIN11 isoforms (Din11L and Din11s) requires specialized approaches:

• Isoform-specific antibodies: Generate antibodies against the unique N-terminal region of Din11L (first 24 amino acids) which does not align with other 2-ODD-C23 homologs .

• SDS-PAGE resolution: Optimize gel conditions to separate the slightly different molecular weights:

  • Use 10-12% polyacrylamide gels with extended run times

  • Din11L will migrate slightly slower than Din11s due to the additional 29 amino acids

• Mass spectrometry:

  • Employ targeted proteomics to identify unique peptides from each isoform

  • Use selected reaction monitoring (SRM) to quantify each isoform separately

• Immunoprecipitation-coupled Western blot:

  • First capture total DIN11 with a pan-specific antibody

  • Then probe with isoform-specific antibodies

• Expression constructs for functional validation:

  • Create constructs expressing either Din11L or Din11s

  • Compare enzymatic activities when expressed in E. coli

  • Din11s shows activity with both homoarginine and arginine, while other isoforms may have different substrate preferences

Sample preparation considerations:
RNA-seq data shows few reads covering the 5′-end of the mRNA but increased coverage 89 bp downstream of the predicted transcription start site, suggesting Din11s may be the predominant isoform under normal conditions .

What experimental controls are essential when using DIN11 antibodies?

When designing experiments with DIN11 antibodies, the following controls are essential:

• Genetic controls:

  • CRISPR/Cas9-generated din11 knockout mutants (negative control)

  • Plants overexpressing Din11L or Din11s (positive control)

  • Mutants of other 2-ODD-C23 family members (At3g49630, At3g50210) to confirm specificity

• Biochemical controls:

  • Pre-absorption of antibody with immunizing peptide (should eliminate signal)

  • Secondary antibody-only control

  • Recombinant DIN11 protein at known concentrations for quantitative analyses

• Sample treatment controls:

  • Unstressed plants vs. plants under conditions known to induce DIN11 (prolonged darkness, wounding, ROS-inducing treatments)

  • Time-course sampling following induction

• Enzyme activity correlation:

  • Measure guanidine and GSA production in parallel with antibody detection

  • Oxygen consumption assays with purified enzyme to correlate protein levels with activity

How should researchers optimize immunohistochemistry protocols for DIN11 detection in plant tissues?

Optimizing immunohistochemistry for DIN11 detection in plant tissues requires careful consideration of fixation, antigen retrieval, and detection methods:

• Tissue preparation and fixation:

  • Fix plant tissues in 4% paraformaldehyde

  • Embed in paraffin and prepare 5 μm thick sections

  • Place on charged slides to prevent tissue loss

• Antigen retrieval optimization:

  • Test multiple methods: citrate buffer (pH 6.0) and EDTA (pH 8.0)

  • Perform heat-induced epitope retrieval at 95°C for approximately 64 minutes

  • Test different retrieval times to determine optimal conditions for DIN11

• Antibody concentration optimization:

  • Perform titration series (0.25–1 μg/mL) to determine optimal antibody concentration

  • Test both overnight incubation at 4°C and shorter incubations at room temperature

• Detection system:

  • For chromogenic detection: Use HRP-conjugated secondary antibodies with DAB development

  • For fluorescence: Use fluorochrome-conjugated secondary antibodies with DAPI counterstain

  • Consider tyramide signal amplification for low-abundance detection

• Image acquisition and quantification:

  • Capture images at 20-100x magnification using appropriate microscopy

  • Use CellProfiler and CellProfiler Analyst for quantitative analysis

  • Apply consistent thresholds across experimental groups

When developing these protocols, pay special attention to potential induction of DIN11 expression by ROS, as these may act as second messengers in response to various stresses .

What are the biochemical considerations when using DIN11 antibodies in activity assays?

When using DIN11 antibodies in conjunction with enzymatic activity assays, researchers should consider several important factors:

• Substrate specificity:

  • DIN11 shows activity with homoarginine as primary substrate

  • Din11s can also use arginine as substrate, though with higher Km (6.0±5.1 mM vs. homoarginine) and lower activity (5.2 ± 0.9 nmol s⁻¹ mg⁻¹)

  • Neither lysine, agmatine, nor other guanidine-containing compounds induce oxygen consumption

• Inhibition patterns:

  • Arginine competitively inhibits Din11s and At3g50210's reaction with homoarginine

  • At3g49630 is insensitive to arginine inhibition

  • Canavanine (arginine antimetabolite) shows competitive inhibition with Din11s but mixed inhibition with At3g50210

• Reaction conditions:

  • Ensure presence of co-factors: Fe²⁺, 2-oxoglutarate, and ascorbate

  • Monitor oxygen consumption during reaction

  • Measure products: guanidine and glutamate semialdehyde/P5C

• Immunoprecipitation considerations:

  • Use antibodies to pull down DIN11 from plant extracts before activity assays

  • Include proper controls to account for potential co-precipitating proteins

  • Wash stringently to remove inhibitors while preserving activity

• Correlation analyses:

  • Compare DIN11 protein levels (by Western blot) with enzyme activity

  • Account for post-translational modifications that may affect activity but not antibody detection

How can researchers utilize DIN11 antibodies to study stress responses in plants?

DIN11 expression is induced by various stress conditions, making DIN11 antibodies valuable tools for studying plant stress responses:

• Stress induction experiments:

  • Dark treatment: Expose plants to prolonged darkness (24-72 hours)

  • Wounding: Mechanical damage to leaves

  • ROS treatment: Apply hydrogen peroxide or methyl viologen

  • Monitor DIN11 protein levels using Western blot at different time points

• Cellular localization during stress:

  • Perform immunohistochemistry on stressed vs. unstressed plants

  • Co-stain with markers for subcellular compartments

  • Quantify changes in localization patterns using image analysis software

• Protein-protein interaction studies:

  • Use DIN11 antibodies for co-immunoprecipitation experiments

  • Identify stress-specific interaction partners by mass spectrometry

  • Validate interactions using reciprocal co-IP or proximity ligation assays

• Metabolite correlation:

  • Measure guanidine and GSA levels in parallel with DIN11 protein levels

  • Analyze correlation between protein abundance and metabolite accumulation

  • Compare wild-type and din11 mutant plants under stress conditions

• Time-course analyses:

  • Monitor DIN11 protein levels at different time points following stress application

  • Compare protein dynamics with transcriptomic data

  • Investigate potential post-transcriptional regulation mechanisms

This approach allows researchers to determine whether DIN11's potential role in defense responses involves direct production of guanidine, AASA, or GSA, which may function in plant-microbe interactions as hypothesized in the literature .