At5g07670 Antibody

What protein does the At5g07670 gene encode and what is its function in Arabidopsis?

The At5g07670 gene locus in Arabidopsis thaliana encodes a protein that functions in cellular pathways related to autophagy and immune responses. Similar to ATG6 (another Arabidopsis protein), it likely participates in essential cellular processes required for plant growth and development. ATG6 is a common and required subunit of the class III phosphatidylinositol 3-kinase (PtdIns3K) lipid kinase complexes, which regulates autophagosome nucleation in Arabidopsis . The protein encoded by At5g07670 may similarly participate in autophagy-related pathways and potentially interact with immune response regulators like NPR1, given the functional similarities observed in related proteins in this family .

How are antibodies against At5g07670 typically generated for research applications?

Antibodies against At5g07670 protein are typically generated through recombinant protein expression systems. The process involves:

• Cloning the At5g07670 gene sequence or specific epitope-containing fragments into an expression vector

• Expressing the protein in bacterial (E. coli), insect, or mammalian cell systems

• Purifying the recombinant protein using affinity chromatography

• Immunizing animals (typically rabbits for polyclonal antibodies or mice for monoclonal antibodies)

• Screening and purifying the resulting antibodies

For monoclonal antibodies, hybridoma technology is employed following similar protocols used for generating antibodies against other plant proteins, such as those described for PD-1 antibody generation, where hybridoma cells are created through fusion of B cells with myeloma cells .

What are the common applications of At5g07670 antibodies in plant molecular biology?

At5g07670 antibodies are primarily used for:

• Protein detection and quantification: Western blotting to detect the protein in plant tissue extracts, similar to techniques used for detecting ATG6 in nuclear and cytoplasmic fractions

• Protein localization: Immunofluorescence microscopy and subcellular fractionation to determine protein localization, following methods similar to those used for ATG6, which was detected in both cytoplasmic and nuclear fractions in Arabidopsis

• Protein-protein interaction studies: Co-immunoprecipitation (Co-IP) assays to identify protein binding partners, similar to methods used to demonstrate ATG6 interaction with NPR1

• Chromatin immunoprecipitation (ChIP): To study potential DNA-binding properties if the protein functions as a transcription factor or associates with chromatin

• Functional studies: Antibody-mediated neutralization of protein function in cell-free systems

How can I validate the specificity of At5g07670 antibodies for immunoprecipitation experiments?

Validating antibody specificity for immunoprecipitation requires multiple approaches:

• Genetic controls: Compare immunoprecipitation results between wild-type plants and At5g07670 knockout/knockdown lines (if viable, as homozygous ATG6 mutants are lethal )

• Epitope-tagged protein expression: Create transgenic plants expressing tagged versions of At5g07670 (e.g., GFP or mCherry fusions as done with ATG6 ) and validate antibody immunoprecipitation against the known tag

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide before immunoprecipitation; specific binding should be competitively inhibited

• Mass spectrometry validation: Analyze immunoprecipitated proteins by mass spectrometry to confirm identity

• Western blot analysis: Perform western blotting on immunoprecipitated samples to confirm the presence of At5g07670 at the expected molecular weight

For example, in similar studies with ATG6-interacting proteins, researchers validated immunoprecipitation specificity by detecting co-precipitated proteins through western blotting with specific antibodies against interaction partners like NPR1 .

What approaches can be used to study At5g07670 protein interactions with NPR1 and other immunity-related proteins?

Based on methodologies used for studying ATG6-NPR1 interactions, several approaches can be employed:

• Yeast two-hybrid (Y2H) screening: To identify potential protein interactions in a heterologous system

• Bimolecular fluorescence complementation (BiFC): To visualize protein interactions in planta by fusing split fluorescent protein fragments to At5g07670 and potential interacting partners

• Co-immunoprecipitation (Co-IP): Using At5g07670 antibodies to pull down the protein complex from plant extracts followed by immunoblotting for potential partners

• Fluorescence resonance energy transfer (FRET): For studying protein interactions in vivo using fluorescently tagged proteins

• Pull-down assays: Using recombinant GST- or His-tagged At5g07670 to identify interacting partners

For example, researchers demonstrated that ATG6 directly interacts with NPR1 using similar techniques, and showed that ATG6 overexpression significantly increased nuclear accumulation of NPR1 . This methodology can be adapted for studying At5g07670 interactions.

How can subcellular localization of At5g07670 be accurately determined using antibody-based approaches?

Accurate subcellular localization requires multiple complementary techniques:

• Immunofluorescence microscopy: Using At5g07670 antibodies with fluorescently labeled secondary antibodies on fixed plant tissue sections

• Subcellular fractionation followed by immunoblotting: Separating nuclear, cytoplasmic, membrane, and other cellular fractions followed by western blotting with At5g07670 antibodies

• Immuno-electron microscopy: For high-resolution localization studies at the ultrastructural level

• Live-cell imaging with fluorescent protein fusions: Complementing antibody-based approaches with transgenic lines expressing At5g07670-GFP/mCherry fusions

• Co-localization studies: Using markers for specific organelles or cellular compartments

Research on ATG6 demonstrates the value of this multi-method approach: "Under SA treatment, ATG6-mCherry and ATG6-GFP were detected in the cytoplasmic and nuclear fractions" and "ATG6 was also detected in the nuclear fraction of UBQ10::ATG6-GFP and UBQ10::ATG6-mCherry overexpressing plants, and SA promoted both cytoplasm and nuclear accumulation of ATG6" .

What are the optimal fixation and antigen retrieval methods for At5g07670 immunohistochemistry in plant tissues?

Optimal fixation and antigen retrieval methods depend on tissue type and specific experimental goals:

• Fixation options:

  • 4% paraformaldehyde for 2-4 hours (preserves structure while maintaining antigenicity)

  • Ethanol:acetic acid (3:1) for RNA/protein co-detection

  • Methanol for membrane proteins

• Antigen retrieval methods:

  • Heat-induced epitope retrieval (HIER): Incubate sections in citrate buffer (pH 6.0) at 95°C for 10-20 minutes

  • Enzymatic retrieval: Using proteases like proteinase K (1-5 μg/ml) for 5-15 minutes

  • Detergent permeabilization: 0.1-0.5% Triton X-100 for increased antibody accessibility

• Tissue sectioning considerations:

  • Paraffin sections (5-8 μm): Provide good morphology but require extensive processing

  • Cryosections (10-15 μm): Better antigen preservation but poorer morphology

  • Vibratome sections (50-100 μm): Good for 3D reconstruction but poorer resolution

• Blocking conditions:

  • 2-5% BSA or normal serum from the species of secondary antibody origin

  • 0.1-0.3% Triton X-100 for permeabilization

  • Incubation for 1-2 hours at room temperature

These protocols should be adapted from methods shown effective for other plant proteins like ATG6 and NPR1 .

What controls should be included when using At5g07670 antibodies for western blotting?

A comprehensive set of controls should include:

• Positive controls:

  • Recombinant At5g07670 protein at known concentrations

  • Extract from plants overexpressing At5g07670

  • Tissues/conditions known to express high levels of the protein

• Negative controls:

  • At5g07670 knockdown/knockout plant extracts (if viable)

  • Pre-immune serum for polyclonal antibodies

  • Isotype control for monoclonal antibodies

  • Secondary antibody only controls

• Specificity controls:

  • Peptide competition assay (pre-incubation of antibody with immunizing peptide)

  • Probing multiple tissues with varying expression levels

  • Detection of tagged At5g07670 with both tag-specific and At5g07670-specific antibodies

• Loading and transfer controls:

  • Housekeeping proteins (e.g., actin, tubulin)

  • Ponceau S staining of membranes

  • Compartment-specific markers for subcellular fractionation studies

For example, similar quality controls were used in research on ATG6, where "endogenous ATG6 is present in both the nucleus and cytoplasm, and SA treatment promotes the accumulation of ATG6 in the nucleus" .

How should At5g07670 antibody dilutions be optimized for different experimental applications?

Antibody dilution optimization should follow a systematic approach:

For each application:

• Test multiple dilutions in parallel

• Include appropriate positive and negative controls

• Quantify signal-to-noise ratio

• Consider time, temperature, and buffer composition variables

• Validate reproducibility with independent antibody lots

Methodological approaches should adapt techniques used in studies of similar plant proteins, such as those described for PD-1 antibodies where blocking assays were performed at 10 μg/ml for 30 minutes at 4°C .

How can nonspecific binding of At5g07670 antibodies be reduced in immunoprecipitation experiments?

Reducing nonspecific binding requires a systematic approach:

• Pre-clearing samples:

  • Incubate lysates with protein A/G beads for 1 hour before adding antibody

  • Pre-adsorb antibodies with irrelevant proteins/tissues

  • Use species-matched pre-immune serum for pre-clearing

• Optimizing wash conditions:

  • Increase salt concentration (150-500 mM NaCl) gradually

  • Add mild detergents (0.1-0.5% NP-40 or Triton X-100)

  • Include protein competitors (0.1-0.5% BSA)

  • Increase washing steps (5-7 washes)

• Buffer optimization:

  • Test different pH conditions (pH 7.0-8.0)

  • Add reducing agents (1-5 mM DTT)

  • Include protein stabilizers (10% glycerol)

  • Add protease and phosphatase inhibitors

• Antibody considerations:

  • Use affinity-purified antibodies

  • Cross-link antibodies to beads to reduce antibody contamination

  • Test monoclonal vs. polyclonal antibodies

Based on techniques used in similar immunoprecipitation studies, researchers could implement washing protocols similar to those used for ATG6 co-immunoprecipitation experiments that successfully demonstrated interaction with NPR1 .

What factors affect the reproducibility of At5g07670 antibody-based western blot results?

Numerous factors can impact reproducibility:

• Sample preparation variables:

  • Extraction buffer composition (detergents, salts, pH)

  • Protease inhibitor cocktail freshness

  • Protein denaturation conditions (temperature, time)

  • Sample storage conditions and freeze-thaw cycles

• Electrophoresis factors:

  • Gel concentration and composition

  • Running buffer freshness and composition

  • Voltage and running time consistency

  • Pre-cast vs. laboratory-prepared gels

• Transfer variables:

  • Transfer method (wet, semi-dry, dry)

  • Transfer time and voltage/current

  • Buffer composition and temperature

  • Membrane type and pre-treatment

• Antibody-related factors:

  • Antibody storage conditions

  • Lot-to-lot variability

  • Age of antibody preparation

  • Secondary antibody selection

• Detection system variables:

  • ECL reagent freshness

  • Exposure time standardization

  • Image acquisition settings

  • Quantification methods

For example, when studying plant nuclear proteins like ATG6, researchers have found that adding phosphatase inhibitors and using freshly prepared nuclear extraction buffers significantly improved reproducibility of western blot results .

How should contradictory results between immunofluorescence and subcellular fractionation of At5g07670 be reconciled?

Reconciling contradictory localization results requires systematic investigation:

• Methodological validation:

  • Compare fixation methods (aldehyde vs. organic solvent)

  • Evaluate antibody accessibility to different cellular compartments

  • Test multiple antibodies targeting different epitopes

  • Assess potential epitope masking in different preparations

• Biological explanations:

  • Consider dynamic localization dependent on cell cycle, stress, or developmental stage

  • Evaluate potential post-translational modifications affecting localization

  • Investigate isoform-specific localization patterns

  • Assess if protein shuttles between compartments, as seen with NPR1

• Complementary approaches:

  • Use fluorescent protein fusions (N- and C-terminal)

  • Employ cell fractionation with marker validation

  • Utilize immuno-electron microscopy for high-resolution localization

  • Perform functional assays to determine compartment-specific activity

• Controls and standards:

  • Include compartment-specific marker proteins

  • Use plants expressing tagged versions of the protein

  • Compare results with closely related proteins

Research on ATG6 localization provides a relevant example: "ATG6 is localized to both cytoplasm and nucleus, and co-localized with NPR1 in the nucleus," demonstrating how multiple approaches can build a complete picture of protein localization .

How can At5g07670 antibodies be used to investigate protein-DNA interactions in chromatin immunoprecipitation (ChIP) experiments?

Optimizing ChIP with At5g07670 antibodies requires specialized considerations:

• Chromatin preparation:

  • Crosslinking optimization (1-2% formaldehyde for 5-15 minutes)

  • Chromatin shearing to optimal fragment size (200-500 bp)

  • Sonication parameters specific to plant tissues

  • Evaluating chromatin quality by agarose gel electrophoresis

• Immunoprecipitation conditions:

  • Pre-clearing chromatin with protein A/G beads

  • Antibody incubation time (overnight at 4°C)

  • Wash buffer stringency progression

  • Elution and crosslink reversal conditions

• Controls and validation:

  • Input chromatin controls

  • IgG negative controls

  • Positive controls (known targets or tagged protein)

  • Evaluation of enrichment by qPCR before sequencing

• Data analysis considerations:

  • Peak calling algorithms

  • Motif discovery tools

  • Integration with RNA-seq data

  • Comparison with known transcription factor binding sites

This methodology would be particularly relevant if At5g07670, like ATG6, is found to localize to the nucleus and potentially influence gene expression related to plant immunity responses .

What novel approaches can be used to study At5g07670 protein dynamics in living plant cells?

Several cutting-edge approaches can be applied:

• Advanced microscopy techniques:

  • Single molecule tracking with photoactivatable fluorescent proteins

  • Fluorescence recovery after photobleaching (FRAP) for mobility analysis

  • Förster resonance energy transfer (FRET) for real-time interaction studies

  • Fluorescence correlation spectroscopy (FCS) for concentration and diffusion measurement

• Protein proximity labeling:

  • BioID or TurboID fusion proteins to identify proximal interacting proteins

  • APEX2 for electron microscopy-compatible proximity labeling

  • Split-BioID for conditional proximity labeling

• Optogenetic approaches:

  • Light-inducible dimerization to control protein interactions

  • Optogenetic degrons for rapid protein depletion

  • Photoswitchable protein tags for super-resolution imaging

• Protein condensate studies:

  • Analysis of potential phase separation behaviors

  • Investigation of similar condensate formation as observed with NPR1 (SINCs - SA-induced NPR1 condensates)

  • In vitro reconstitution of condensates with purified proteins

Researchers studying NPR1 discovered that "ATG6 can stabilize NPR1 and promote the formation of SINCs (SA-induced NPR1 condensates)-like condensates" , suggesting similar approaches could be valuable for At5g07670 studies.