DGAT3 Antibody

What is DGAT3 and why is it significant in lipid metabolism research?

DGAT3 (Diacylglycerol Acyltransferase 3) represents a distinct class of enzymes involved in triacylglycerol (TAG) synthesis. Unlike DGAT1 and DGAT2, which are integral membrane proteins typically located in the endoplasmic reticulum, DGAT3 enzymes are described as soluble proteins with unique structural features .

In Arabidopsis thaliana, DGAT3 has been characterized as a [2Fe-2S] protein that exhibits DGAT activity in vitro, making it the first metalloprotein described as a DGAT . This distinctive feature has significant implications for understanding alternative pathways of TAG synthesis, particularly in plants.

Recent research has shown that DGAT3 plays important roles in various biological contexts:

• In Chlamydomonas reinhardtii, DGAT3 appears to participate in a soluble TAG synthesis pathway in the chloroplast

• In tung tree (Vernicia fordii), DGAT3 expression varies across different tissues, with highest expression observed in flowers compared to seeds

• In Arabidopsis, DGAT3 has been implicated in powdery mildew interactions, with evidence suggesting pathogen exploitation of host plastoglobuli functions via DGAT3

How does DGAT3 structurally differ from other DGAT family members?

DGAT3 exhibits several structural features that distinguish it from DGAT1 and DGAT2:

• Hydrophobicity profile: The content of hydrophobic regions in DGAT3 is considerably lower than those of DGAT1 and DGAT2. While DGAT1 and DGAT2 contain multiple hydrophobic regions that translate into transmembrane segments, DGAT3 appears to have no transmembrane domains .

• Catalytic motifs: In DGAT1 and DGAT2, the catalytic motifs either flank or are partially embedded in hydrophobic regions, whereas the putative catalytic motifs of C. reinhardtii DGAT3 are flanked by hydrophilic regions .

• Iron-sulfur cluster: A distinctive feature of DGAT3 is the presence of a [2Fe-2S] cluster-binding domain. In C. reinhardtii DGAT3, this domain was modeled on the thioredoxin-like ferredoxin of Aquifex aeolicus . This makes DGAT3 unique as the first metalloprotein described with DGAT activity .

• Conserved residues: DGAT3s are distinct from DGAT1s and DGAT2s because none of the completely conserved residues in DGAT1s (41 residues) and DGAT2s (16 residues) are found in DGAT3 sequences .

• Domain structure: In C. reinhardtii, DGAT3 features a transit peptide of approximately 70 amino acids, followed by a disordered region of about 223 amino acids containing putative acyltransferase motifs, and then the 2Fe-2S cluster-binding domain .

What challenges are typically encountered when producing antibodies against DGAT3?

Researchers face several significant challenges when producing antibodies against DGAT3:

• Protein instability: As demonstrated with Arabidopsis thaliana DGAT3, the recombinant purified protein produced from E. coli is very unstable in vitro . This instability complicates the production of high-quality antigens for antibody generation.

• Iron-sulfur cluster: The presence of a [2Fe-2S] cluster adds complexity to protein folding and stability. AtDGAT3 is unstable in its reduced form, which affects the consistency of the antigen preparation .

• Expression difficulties: Full-length DGAT3 proteins have shown limited activity in vitro compared to truncated versions. For instance, a shorter protein version of AtDGAT3 devoid of its N-terminal putative chloroplast transit peptide (Δ46AtDGAT3) demonstrated greater stability in vitro, allowing for biochemical and spectroscopic characterization .

• Lack of established protocols: Unlike DGAT1 and DGAT2, for which antibody production protocols have been developed and optimized over time, DGAT3-specific antibody production remains less standardized, as evidenced by the limited literature on DGAT3 antibodies compared to other DGAT family members .

• Specificity concerns: The distinct structure of DGAT3 compared to other family members requires careful epitope selection to ensure antibody specificity and avoid cross-reactivity with other proteins containing [2Fe-2S] clusters.

What are the recommended approaches for DGAT3 antigen preparation?

Based on successful approaches with other DGAT family members and the specific challenges of DGAT3, the following methods are recommended:

• Recombinant protein expression systems:

  • Expression in E. coli has been successful for generating antigens for DGAT family members, with purification via immobilized-metal affinity chromatography

  • For DGAT3, consider using truncated constructs lacking the transit peptide (similar to Δ46AtDGAT3 or Δ75AtDGAT3), which have shown improved stability in vitro

• Fusion protein strategies:

  • MBP-fusion proteins have proven effective for DGAT2 antigen production (MBP-DGAT2)

  • For DGAT3, similar fusion approaches may help improve solubility and stability

• Peptide antigens:

  • Synthetic peptides corresponding to unique regions of DGAT3 can be designed for generating antibodies

  • Target hydrophilic regions of DGAT3 that are predicted to be surface-exposed and avoid regions with the [2Fe-2S] cluster

• Purification protocol optimization:

  • Include reducing agents during purification to maintain the [2Fe-2S] cluster integrity

  • Consider rapid purification procedures under anaerobic conditions to prevent oxidative damage

  • Implement buffer optimization to maximize protein stability during purification

The success of the DGAT2 antibody production strategy described in search result provides a useful template:

"The antigen rDGAT2 was purified from overexpressed E. coli as reported and concentrated with a Centricon-10 concentrator to a protein concentration of 1 mg/ml. Anti-rDGAT2 serum was produced in rabbits immunized with the purified rDGAT2 fusion protein."

How can researchers validate the specificity of DGAT3 antibodies?

Validating antibody specificity is critical, especially for less-studied proteins like DGAT3. The following validation approaches are recommended:

• Western blot analysis:

  • Compare wild-type samples with DGAT3 knockout/knockdown lines

  • Include recombinant DGAT3 protein as a positive control

  • Test for cross-reactivity with DGAT1 and DGAT2 proteins

• Immunoprecipitation followed by mass spectrometry:

  • This approach was successfully used for AtDGAT3, where "In-gel tryptic digestion was performed with the Progest system according to after protein reduction (10 mM DTT) and alkylation (55 mM iodoacetamide). NanoLC-MS/MS analysis was performed using an Ultimate 3000 LC system connected to a LTQ Orbitrap mass spectrometer"

• Immunofluorescence with controls:

  • Compare staining patterns in tissues with known DGAT3 expression patterns

  • Include appropriate negative controls (pre-immune serum, isotype controls)

  • Perform peptide competition assays where available

• RNA expression correlation:

  • Verify that antibody detection correlates with mRNA expression patterns

  • In tung tree tissues, DGAT3 mRNA was highly expressed in flowers compared to seeds during oil accumulation , providing a useful reference for validation

• Subcellular localization confirmation:

  • For C. reinhardtii DGAT3, chloroplast localization was predicted

  • For Arabidopsis DGAT3, localization to plastoglobuli was observed under specific conditions

What optimization strategies are effective for DGAT3 immunodetection?

Given the unique properties of DGAT3, standard immunodetection protocols may require optimization:

• Western blot optimization:

  • Buffer systems: Consider using native PAGE for maintaining protein structure, especially for preserving the [2Fe-2S] cluster

  • Reducing conditions: Test both reducing and non-reducing conditions as they may affect epitope accessibility

  • Transfer conditions: Optimize transfer parameters for soluble proteins

• Immunohistochemistry considerations:

  • Fixation methods: Compare cross-linking fixatives (paraformaldehyde) vs. precipitating fixatives (acetone)

  • Antigen retrieval: Test different antigen retrieval methods, particularly for formalin-fixed tissues

  • Signal amplification: Consider tyramide signal amplification for low-abundance proteins

• Immunofluorescence optimization:

  • For DGAT3 localization to plastoglobuli, specialized protocols may be needed

  • As demonstrated for DGAT3-GFP detection: "Immunoblotting of total leaf protein extracts using an anti-GFP antibody confirmed the accumulation of DGAT3-GFP at 10 dpi in response to powdery mildew infection"

• Sample preparation considerations:

  • Protein extraction buffers should be optimized to preserve DGAT3 stability

  • Include protease inhibitors and reducing agents during extraction

  • Consider detergent selection carefully, as DGAT3 is a soluble protein unlike DGAT1/2

How can DGAT3 antibodies be utilized to study protein-protein interactions?

DGAT3 likely functions within protein complexes, and antibodies can help elucidate these interactions:

• Co-immunoprecipitation approaches:

  • Use DGAT3 antibodies for pull-down experiments followed by mass spectrometry

  • For membrane-associated pools of DGAT3, consider crosslinking prior to immunoprecipitation

  • Include appropriate controls to distinguish specific from non-specific interactions

• Proximity labeling techniques:

  • BioID or APEX2 fusions with DGAT3 combined with antibody detection can reveal proximity interactors

  • These approaches are particularly useful for transient interactions

• Two-hybrid system validation:

  • Putative interactions identified in yeast or bacterial two-hybrid systems can be validated using co-immunoprecipitation with DGAT3 antibodies

• FRET/FLIM analysis:

  • Combine fluorescently-tagged proteins with antibody detection to study interactions in situ

  • This can be particularly useful for studying DGAT3 interactions in plastoglobuli

Research has suggested that DGAT3 may interact with other proteins involved in TAG synthesis pathways. In the case of powdery mildew interactions, evidence suggests that "DGAT3 is localized to PGs formed in response to powdery mildew or dark induction" and that "DGAT3 is unusual in that it is a [2Fe-2S] metalloenzyme that is unstable in its reduced form" , indicating potential interactions with proteins that regulate redox status.

How can researchers address the challenges of studying DGAT3 post-translational modifications?

DGAT3 activity and stability may be regulated by post-translational modifications (PTMs), which can be studied using antibodies:

• Phosphorylation analysis:

  • Use phospho-specific antibodies if phosphorylation sites are known

  • Combine immunoprecipitation with phospho-proteomic analysis

  • Compare PTM status under different physiological conditions

• Redox state detection:

  • Given that DGAT3 contains a [2Fe-2S] cluster and "is unstable in its reduced form" , redox state-specific antibodies might be valuable

  • Alternatively, combine immunoprecipitation with redox proteomics approaches

• Stability and turnover studies:

  • Use DGAT3 antibodies to track protein abundance under different conditions

  • Evidence suggests that "DGAT3 is unusual in that it is a [2Fe-2S] metalloenzyme that is unstable in its reduced form. Thus, localized PG redox status could have a profound influence on DGAT3 stability"

• Protein complex formation:

  • Blue native PAGE combined with immunoblotting can reveal different DGAT3-containing complexes

  • This approach is particularly relevant given the potential for DGAT3 to participate in multiprotein complexes

What strategies can be employed for analyzing DGAT3 under different stress conditions?

DGAT3 has been implicated in stress responses, making stress-specific analysis important:

• Differential expression analysis:

  • Compare DGAT3 protein levels across stress conditions using quantitative immunoblotting

  • In C. reinhardtii, "dgat3 mRNAs increased 2-fold as early as 1 h into the light period, peaked at about 12 h (an average value of about 4-fold) and remained elevated until the end of the experiment (3-fold)"

• Subcellular localization changes:

  • Track DGAT3 localization under different stress conditions

  • For example, in Arabidopsis, "We only observe DGAT3-GFP localized in PGs formed in response to powdery mildew or dark induction"

• Activity correlation studies:

  • Correlate DGAT3 protein levels with TAG accumulation under stress

  • In C. reinhardtii, "TAGs accumulated in cc-4348 upon transferring the cells to light, reaching a maximum intensity at 6 h into the light period and decreasing considerably at 24 h"

• Protein-protein interaction changes:

  • Examine how stress affects DGAT3 interactome using co-immunoprecipitation

  • This is particularly relevant given evidence that "Golor4_2520209 could also promote the recruitment and stability of DGAT3 through its influence on FBN2 interactors"

The table below summarizes observed DGAT3 responses to different stress conditions:

What are the primary causes of false-positive and false-negative results with DGAT3 antibodies?

Several factors can contribute to misleading results when working with DGAT3 antibodies:

Causes of false positives:

• Cross-reactivity with other [2Fe-2S] proteins:

  • The distinctive [2Fe-2S] cluster domain in DGAT3 shares structural similarities with other iron-sulfur proteins

  • Validate specificity against known [2Fe-2S] proteins in your experimental system

• Non-specific binding to hydrophobic domains:

  • Even though DGAT3 is more soluble than other DGAT family members, it may still contain hydrophobic regions that can lead to non-specific antibody binding

  • Include appropriate blocking agents and detergents in immunodetection protocols

• Reactivity with bacterial contaminants:

  • E. coli proteins co-purifying with recombinant DGAT3 may generate antibodies that cross-react with bacterial proteins

  • Perform careful purification of antigens and validate antibodies against multiple negative controls

Causes of false negatives:

• Protein instability:

  • The documented instability of DGAT3, particularly in its reduced form, may lead to protein degradation during sample preparation

  • "AtDGAT3 is unusual in that it is a [2Fe-2S] metalloenzyme that is unstable in its reduced form"

• Epitope masking:

  • The [2Fe-2S] cluster or interacting proteins may mask antibody epitopes

  • Test different extraction and denaturation conditions

• Low abundance:

  • DGAT3 expression levels vary significantly across tissues and conditions

  • In tung tree, "DGAT3 was the most abundant mRNA in tung leaves, flowers and immature seeds prior to oil accumulation" but levels may be much lower in other contexts

• Post-translational modifications:

  • PTMs may alter epitope recognition

  • Consider using multiple antibodies targeting different regions of DGAT3

How can researchers compare the efficacy of different antibody detection methods for DGAT3?

When evaluating different detection methods for DGAT3, consider the following comparative approach:

• Western blot vs. ELISA:

  • Western blotting provides size information and can distinguish specific from non-specific binding

  • ELISA offers higher throughput and potential quantification but may be more prone to cross-reactivity

• Immunofluorescence vs. immunohistochemistry:

  • Immunofluorescence provides higher resolution for subcellular localization

  • Immunohistochemistry may offer better tissue context and is more stable long-term

• Direct comparison methodology:

  • Use the same samples across different detection methods

  • Include appropriate positive and negative controls for each method

  • Quantify and statistically analyze the results when possible

• Method-specific optimization:

  • For Western blotting: Test different extraction buffers, gel types, and transfer conditions

  • For immunolocalization: Compare fixation methods, permeabilization approaches, and detection systems

The efficacy of fluorescence anisotropy for studying DGAT3 interactions with lipids has been demonstrated: "Steady-state anisotropy from DGAT3 as a function of [lipid]/[protein] ratio where the lipid is POPC (blue) and POPG (purple). Excitation and emission wavelengths were 290 and 340 nm, respectively" . This approach could be complemented with antibody-based methods for comprehensive analysis.

What reference materials should be included when validating novel DGAT3 antibodies?

Proper validation of DGAT3 antibodies requires thoughtfully selected reference materials:

• Positive controls:

  • Recombinant DGAT3 protein (full-length and relevant fragments)

  • Tissues with confirmed high DGAT3 expression (e.g., flowers in tung tree, which showed highest expression levels compared to seeds )

  • Transgenic systems overexpressing DGAT3

• Negative controls:

  • DGAT3 knockout or knockdown lines

  • Pre-immune serum controls

  • Peptide competition assays

• Specificity controls:

  • Recombinant DGAT1 and DGAT2 proteins to verify absence of cross-reactivity

  • Other [2Fe-2S] proteins to confirm specificity against the structural domain

• Technical validation samples:

  • Standard curves with known amounts of purified protein

  • Samples from multiple biological replicates

  • Different tissue types with varying DGAT3 expression levels

• Inter-laboratory validation materials:

  • Standard reference samples that can be shared between research groups

  • Detailed protocols for reproducible results across laboratories