NTMC2T6.1 Antibody

What is NTMC2T6.1 and why is it important for plant research?

NTMC2T6.1 (AT1G53590) is a plant-exclusive SMP (Synaptotagmin-like Mitochondrial-lipid binding Proteins) domain protein that has not been extensively studied to date . The protein is significant because it represents a class of proteins potentially involved in lipid transfer between cellular membranes in plants. NTMC2T6.1 has been identified in highly purified vacuoles from mature leaves by 1-D SDS-PAGE LC MS/MS, although it was absent in 2-D LC MS/MS analyses of the same fractions . Notably, the protein shows increased phosphorylation following flg22 elicitor treatment in Arabidopsis cell cultures, suggesting potential roles in plant immune responses .

Research on NTMC2T6.1 is particularly important because membrane contact sites (MCS) play critical roles in cell biology, yet knowledge about proteins facilitating lipid transfer between organelles in plants remains limited. Understanding NTMC2T6.1 function could provide significant insights into lipid trafficking between the endoplasmic reticulum and other organelles, including the trans-Golgi network.

What are the structural characteristics of NTMC2T6.1?

In-silico analyses have revealed that NTMC2T6.1 shares common structural elements with SYT6 (Synaptotagmin 6). The protein is characterized by:

• Two short transmembrane (TM) domains in the N-terminal region

• An SMP domain followed by a single C2 domain

• A predicted disordered domain

• One helix coiled-coil domain in AtNTMC2T6.2 (a related protein)

The transmembrane domains anchor the protein to the endoplasmic reticulum, while the SMP domain is likely involved in lipid transfer between membranes. The C2 domain may function in calcium-dependent membrane binding or protein-protein interactions, although this remains to be experimentally confirmed for NTMC2T6.1 specifically.

Where is NTMC2T6.1 localized in plant cells?

Transient co-expression studies in Nicotiana benthamiana have demonstrated that the transmembrane domain of AtNTMC2T6.1 (M1-R73, AtNTMC2T6.1 TM-GFP) perfectly co-localizes with ER markers, indicating that these are ER-anchored proteins . Confocal microscopy analyses revealed a reticulated pattern characteristic of ER localization.

Interestingly, while AtNTMC2T6.1 is localized to the ER, it also appears at specific contact sites between the ER and trans-Golgi network (TGN). Studies showed high colocalization with:

• VAMP721 (a TGN protein)

• AtSYT6 (another ER-localized protein)

• AtTEX2B (an ER protein)

These findings suggest that NTMC2T6.1 is specifically enriched at ER-TGN contact sites, potentially participating in lipid exchange between these membrane compartments.

What protein interactions has NTMC2T6.1 been shown to participate in?

Co-immunoprecipitation (Co-IP) assays have revealed several protein interactions involving NTMC2T6.1:

Notably, NTMC2T6.1 did not associate with SYP41, a Qa-SNARE protein at TGN vesicles, suggesting specificity in its interactions with TGN components . The interaction data indicates that NTMC2T6.1 likely forms part of a protein complex at ER-TGN contact sites along with SYT6, TEX2B, and specific SNARE proteins.

How can researchers generate specific antibodies against NTMC2T6.1?

Developing specific antibodies against NTMC2T6.1 requires careful epitope selection and validation strategies:

• Epitope selection: For optimal specificity, target unique regions of NTMC2T6.1 not shared with homologous proteins like NTMC2T6.2. The SMP domain or the linker region between the SMP and C2 domains often contain unique sequences suitable for antibody generation.

• Antigen preparation methods:

  • Recombinant protein expression: Express full-length or domain-specific fragments in bacterial systems

  • Synthetic peptides: Design peptides (15-25 amino acids) from unique regions

• Immunization and screening approach:

  • Immunize rabbits with the purified antigen following standard protocols

  • Screen antibody specificity against both the immunizing antigen and the native protein

  • Include cross-reactivity tests against related SMP proteins

When designing antibodies, researchers should consider that the binding specificity is determined by the large interaction surface between antibodies and their antigens . Based on insights from other antibody design studies, creating highly specific antibodies often requires identifying "hot-spot" residues that contribute significantly to binding .

What approaches can validate the specificity of NTMC2T6.1 antibodies?

Validating antibody specificity for NTMC2T6.1 requires a multi-faceted approach:

• Western blot analysis:

  • Test against wild-type plant tissue, knockout/knockdown lines, and plants overexpressing NTMC2T6.1

  • A specific antibody should detect a single band of the expected molecular weight in wild-type and overexpression samples, but not in knockout lines

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP from plant tissue lysates and identify pulled-down proteins by MS

  • NTMC2T6.1 should be among the most abundant proteins identified

• Immunofluorescence microscopy:

  • Compare localization patterns with GFP-tagged NTMC2T6.1

  • Pattern should match the expected ER reticulated pattern with enrichment at TGN contact sites

• Antigen competition assay:

  • Pre-incubate antibody with purified antigen before immunodetection

  • Signal should be blocked in proportion to antigen concentration

• Knockout/knockdown controls:

  • Generate CRISPR/Cas9 knockout or RNAi knockdown plants

  • Validate absence or reduction of signal in these controls

These validation approaches align with established antibody validation methods in the field and ensure reliable experimental results.

How do post-translational modifications affect NTMC2T6.1 antibody recognition?

Post-translational modifications (PTMs) can significantly impact antibody recognition of NTMC2T6.1:

• Phosphorylation effects:

  • NTMC2T6.1 has been shown to undergo increased phosphorylation after flg22 elicitor treatment

  • Antibodies raised against unmodified peptides may show reduced binding to phosphorylated forms

  • Consider generating modification-specific antibodies if studying phosphorylation events

• Strategic considerations:

  • Generate phospho-specific antibodies when studying immune responses

  • Avoid epitopes containing predicted phosphorylation sites for general detection

  • Use multiple antibodies targeting different regions for complete protein analysis

• Experimental approach:

  • Test antibody recognition using lambda phosphatase-treated and untreated samples

  • Compare detection before and after flg22 treatment to assess phosphorylation impact

  • Evaluate antibody binding to synthetic peptides with and without phosphorylation

For comprehensive analysis of NTMC2T6.1 during signaling events, researchers should consider developing both phosphorylation-dependent and phosphorylation-independent antibodies, similar to approaches used for other signaling proteins in antibody-based assays .

What are optimal protocols for detecting NTMC2T6.1 via Western blotting?

For successful Western blot detection of NTMC2T6.1, consider the following optimized protocol:

• Sample preparation:

  • Extract proteins from plant tissues using buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail

  • Include 1 mM PMSF and phosphatase inhibitors if studying phosphorylated forms

  • Heat samples at 70°C (not 95°C) for 10 minutes to minimize aggregation of membrane proteins

• Gel electrophoresis conditions:

  • Use 10% SDS-PAGE for optimal resolution of NTMC2T6.1 (predicted MW: ~52 kDa)

  • Load appropriate positive controls (e.g., GFP-tagged NTMC2T6.1 from transient expression)

• Transfer parameters:

  • Wet transfer at 30V overnight at 4°C to ensure complete transfer of membrane-associated proteins

  • Use PVDF membrane (0.45 μm pore size) pre-activated with methanol

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature

  • Incubate with primary antibody (1:1000) in 1% BSA in TBST overnight at 4°C

  • Wash extensively (4 × 10 minutes) with TBST

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

• Detection strategy:

  • Use enhanced chemiluminescence (ECL) detection system

  • For low abundance detection, consider using ECL Prime or other high-sensitivity substrates

  • Expected band size: approximately 52 kDa

This protocol incorporates methodological principles from standard immunoblotting techniques adapted for membrane-associated plant proteins.

How can I optimize co-immunoprecipitation for studying NTMC2T6.1 interactions?

Optimized co-immunoprecipitation protocol for NTMC2T6.1 interaction studies:

• Cell lysis optimization:

  • Use a gentle lysis buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40 or 0.5% digitonin, 10% glycerol, 1 mM EDTA, and protease inhibitor cocktail

  • Include 1 mM DTT to maintain protein structure

  • Lyse cells on ice for 30 minutes with gentle mixing

• Pre-clearing strategy:

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C

  • Remove non-specific binding proteins by centrifugation (14,000 × g, 10 minutes)

• Immunoprecipitation conditions:

  • Incubate pre-cleared lysate with anti-NTMC2T6.1 antibody (2-5 μg) overnight at 4°C with gentle rotation

  • Add 30 μl of Protein A/G beads and incubate for additional 3 hours

  • Wash beads 4-5 times with wash buffer (lysis buffer with reduced detergent concentration)

• Protein complex elution:

  • Elute bound proteins with 2× SDS sample buffer at 70°C for 10 minutes

  • Alternatively, use native elution with excess antigen peptide for downstream functional assays

• Analysis of interaction partners:

  • Analyze by SDS-PAGE followed by western blotting for known/suspected partners

  • For unbiased discovery, submit samples for mass spectrometry analysis

This protocol has been shown to effectively isolate NTMC2T6.1-containing complexes including AtSYT6, AtTEX2B, VAMP721, and VAMP727 in previous studies , providing a solid foundation for exploring novel interaction partners.

What microscopy techniques are most suitable for visualizing NTMC2T6.1 localization?

For optimal visualization of NTMC2T6.1 localization, consider these microscopy approaches:

• Confocal laser scanning microscopy:

  • Primary technique for visualizing ER-TGN contact sites

  • Use Airyscan or similar super-resolution confocal methods for resolving contact sites

  • Multi-channel imaging to co-visualize NTMC2T6.1 with ER markers (e.g., mCherry-HDEL) and TGN markers (e.g., VHA-a1-mRFP)

• Sample preparation methods:

  • For immunofluorescence: Fix samples with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • For live-cell imaging: Express fluorescently tagged NTMC2T6.1 (C-terminal tag preferred to avoid TM domain disruption)

• Advanced techniques for detailed analyses:

  • FRET (Förster Resonance Energy Transfer) to study protein-protein interactions in vivo

  • FRAP (Fluorescence Recovery After Photobleaching) to analyze protein dynamics at contact sites

  • 3D-SIM (Structured Illumination Microscopy) for enhanced resolution of membrane contact sites

• Quantification approaches:

  • Measure Pearson's correlation coefficient for colocalization analyses

  • Count and measure contact site numbers and sizes

  • Track temporal changes in response to stimuli or stress conditions

Previous imaging studies have successfully used these approaches to visualize NTMC2T6.1 at ER-TGN contact sites, showing it as a reticulated pattern perfectly co-localizing with ER markers while also showing contact points with TGN markers .

How can antibodies help determine the orientation of NTMC2T6.1 in the ER membrane?

Determining membrane protein topology requires specialized antibody-based approaches:

• Epitope accessibility assay:

  • Generate antibodies against epitopes in different domains of NTMC2T6.1

  • Perform immunofluorescence with and without membrane permeabilization

  • Epitopes accessible without permeabilization face the cytosol

  • Epitopes requiring permeabilization face the ER lumen

• Protease protection assay with immunodetection:

  • Isolate microsomes containing NTMC2T6.1

  • Treat with proteases (e.g., trypsin, proteinase K) with or without detergent

  • Analyze protected fragments using domain-specific antibodies

  • Cytosolic domains will be digested while luminal domains remain protected

• Experimental design considerations:

  • Create a panel of antibodies targeting:

    • N-terminal region (before first TM domain)

    • Loop between TM domains

    • SMP domain

    • C2 domain

    • C-terminal region

  • Use GFP-fusion constructs with known topology as positive controls

• Data interpretation framework:

Based on in silico predictions and experimental evidence from related proteins, NTMC2T6.1 likely has both N- and C-termini facing the cytosol, with its SMP domain positioned to transfer lipids at membrane contact sites .

What strategies can be employed to study the lipid transfer function of NTMC2T6.1?

Investigating NTMC2T6.1's lipid transfer function requires specialized approaches:

• In vitro lipid transfer assays:

  • Express and purify the SMP domain of NTMC2T6.1

  • Prepare donor liposomes containing fluorescent lipids

  • Measure transfer of fluorescent lipids to acceptor liposomes

  • Compare with known SMP proteins as positive controls

• Cellular lipid analysis:

  • Generate NTMC2T6.1 knockout/knockdown plants

  • Compare lipid composition of isolated ER and TGN membranes using lipidomics

  • Analyze changes in glycerolipids, ceramides, and phospholipids

  • Monitor recovery after complementation with wild-type NTMC2T6.1

• Structure-function analysis using antibodies:

  • Generate antibodies against the SMP domain

  • Test inhibition of lipid transfer activity in vitro

  • Map critical residues by testing mutant proteins

  • Use antibodies to immunoprecipitate active complexes

• Lipid binding assays:

  • Perform lipid overlay assays to determine lipid binding specificity

  • Use purified SMP domain and test various lipid species

  • Quantify binding affinity using surface plasmon resonance

  • Compare results with other plant SMP proteins

Based on studies of related proteins, NTMC2T6.1 may be involved in transferring specific glycerolipids between the ER and TGN, similar to the role of AtSYT1 or AtSYT3 in transferring glycerolipids at other membrane contact sites .

How does NTMC2T6.1 function compare to other plant SMP domain-containing proteins?

Comparative analysis of plant SMP proteins reveals functional specialization:

Research approaches to compare these proteins:

• Comparative antibody-based proteomics:

  • Immunoprecipitate each SMP protein and analyze interactomes

  • Identify common and unique interactors

  • Map protein complex networks at different contact sites

• Functional complementation tests:

  • Express NTMC2T6.1 in plants lacking other SMP proteins

  • Test for rescue of phenotypes

  • Analyze domain swapping experiments to identify functional regions

• Evolutionary analysis with immunodetection validation:

  • Analyze SMP proteins across plant species

  • Test cross-reactivity of antibodies

  • Correlate evolutionary conservation with functional importance

NTMC2T6.1 appears to be specialized for ER-TGN contact sites, whereas other plant SMP proteins function at different membrane interfaces. This specialization likely evolved to manage distinct lipid transfer requirements between various organelles .

How can antibodies be used to study NTMC2T6.1 dynamics during stress responses?

Investigating NTMC2T6.1 stress dynamics using antibody-based approaches:

• Stress-induced phosphorylation analysis:

  • Generate phospho-specific antibodies targeting known/predicted phosphorylation sites

  • Expose plants to stresses (pathogen elicitors, abiotic stressors)

  • Perform western blots with phospho-specific and total NTMC2T6.1 antibodies

  • Calculate phosphorylation ratios across stress time courses

• Quantitative changes in protein localization:

  • Perform immunofluorescence before and after stress application

  • Quantify changes in ER-TGN contact site number and NTMC2T6.1 enrichment

  • Correlate with functional outcomes (e.g., lipid composition changes)

• Stress-induced protein complex remodeling:

  • Conduct co-immunoprecipitation under control and stress conditions

  • Identify stress-specific interaction partners

  • Monitor changes in established interactions (e.g., with VAMP721)

• Experimental design for flg22 treatment studies:

Given that NTMC2T6.1 shows increased phosphorylation after flg22 elicitor treatment , it likely plays a role in plant immune responses, potentially by modifying lipid transfer between the ER and TGN during infection. Antibody-based approaches are ideal for tracking these dynamic changes with high temporal resolution.

How can researchers troubleshoot non-specific binding of NTMC2T6.1 antibodies?

When encountering non-specific binding with NTMC2T6.1 antibodies, implement these troubleshooting strategies:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, casein, non-fat dry milk)

  • Increase blocking time (2-3 hours at room temperature)

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

• Antibody dilution series:

  • Test primary antibody dilutions ranging from 1:500 to 1:5000

  • Optimize secondary antibody dilutions (typically 1:2000 to 1:10000)

  • Consider secondary antibodies with reduced cross-reactivity to plant proteins

• Pre-absorption protocol:

  • Incubate antibody with plant extract from knockout lines

  • Remove antibodies binding to non-specific targets

  • Use pre-absorbed antibody for improved specificity

• Sample preparation modifications:

  • Adjust lysis conditions to reduce protein aggregation

  • Consider native vs. denaturing conditions based on epitope accessibility

  • Use freshly prepared samples to minimize degradation products

The techniques for improving antibody specificity align with established methods in the antibody field , adapted specifically for plant membrane proteins like NTMC2T6.1.

How can researchers resolve contradictory data about NTMC2T6.1 subcellular localization?

When facing contradictory localization data, employ these methodological approaches:

• Multi-technique validation strategy:

  • Compare results from different detection methods:

    • Fluorescent protein tagging (N- vs. C-terminal)

    • Immunofluorescence with different fixation protocols

    • Biochemical fractionation with immunoblotting

    • Electron microscopy with immunogold labeling

• Control experiments to resolve discrepancies:

  • Verify tag interference: Compare N-terminal, C-terminal, and internal tags

  • Test expression levels: Compare endogenous vs. overexpression systems

  • Validate in multiple plant species and tissue types

  • Use tagged known markers of relevant compartments as controls

• Potential explanations for contradictory results:

Given the evidence that NTMC2T6.1 localizes to specific ER-TGN contact sites , contradictory data might reflect challenges in visualizing these discrete contact points rather than broad distribution patterns. Advanced imaging techniques like 3D-SIM or STORM microscopy may help resolve these discrepancies.

What are emerging antibody-based technologies for studying NTMC2T6.1 function?

Cutting-edge approaches for NTMC2T6.1 functional studies include:

• Proximity labeling with antibody validation:

  • Express NTMC2T6.1 fused to BioID or APEX2

  • Identify proteins in close proximity at ER-TGN contact sites

  • Validate key interactions with co-IP using specific antibodies

  • Map the spatial organization of contact site proteins

• Antibody-based protein engineering:

  • Generate intrabodies (intracellular antibodies) against NTMC2T6.1

  • Express in plants to disrupt specific interactions or functions

  • Create domain-specific inhibitory antibodies

  • Validate phenotypes against knockout/knockdown lines

• Single-molecule tracking:

  • Use fluorescently labeled Fab fragments against NTMC2T6.1

  • Track protein dynamics at contact sites in living cells

  • Analyze diffusion rates and confinement zones

  • Correlate with functional changes during stress responses

• Structural biology applications:

  • Use antibodies to stabilize NTMC2T6.1 for crystallization

  • Employ cryo-EM techniques with antibody fragments

  • Develop nanobodies against specific conformational states

  • Compare structural details with other SMP proteins

Antibody design principles, including the identification of hot-spot residues and specificity determination , will be critical for developing these advanced applications for NTMC2T6.1 research.