What is the At1g78820 gene and its function in Arabidopsis thaliana?

At1g78820 is a gene in the model plant Arabidopsis thaliana (Mouse-ear cress) that encodes a protein involved in cellular processes. The gene product has been studied in the context of the unfolded protein response (UPR), which is a cellular stress response related to the endoplasmic reticulum. When proteins fail to fold properly in the ER, the UPR is activated, leading to regulation of various genes including potential involvement of At1g78820. The gene has been identified as part of gene expression analysis studies using Affymetrix GeneChips, particularly in experiments examining cellular responses to ER stress induced by agents such as tunicamycin and DTT. The protein encoded by At1g78820 may function within the secretory system, potentially as part of the cell's mechanism to improve protein folding and transport, degrade unwanted proteins, or regulate secretory protein entry into the ER .

What are the recommended applications for the At1g78820 antibody?

The At1g78820 antibody is primarily recommended for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blot (WB) applications for the identification of the target antigen. These techniques allow researchers to detect and quantify the At1g78820 protein in various experimental settings. For Western Blot applications, the antibody enables visualization of the protein based on molecular weight following gel electrophoresis and membrane transfer. ELISA applications permit quantitative analysis of the protein in solution. The antibody has been specifically validated for these applications in Arabidopsis thaliana samples. Researchers should ensure proper experimental controls are included to validate specificity, such as positive controls from known Arabidopsis tissues expressing the protein and negative controls using samples where the protein is absent or blocked. It's important to note that the antibody is designated for research use only and should not be employed in diagnostic or therapeutic procedures .

What are the optimal storage conditions for maintaining At1g78820 antibody activity?

For optimal preservation of At1g78820 antibody activity, the product should be stored at -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can significantly diminish antibody functionality through protein denaturation and aggregation. The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative. This formulation helps maintain stability during storage. For short-term use (within one week), the antibody can be stored at 4°C, but long-term storage requires freezing conditions. When handling the antibody, it's advisable to aliquot the stock solution into smaller volumes based on anticipated usage to minimize freeze-thaw cycles. Each aliquot should be properly labeled with the antibody identification, date of aliquoting, and any dilution information. Prior to experimental use, allow the antibody to equilibrate to room temperature completely before opening the vial to prevent condensation that could introduce contaminants .

How should researchers prepare and optimize Western blot protocols for At1g78820 antibody?

When preparing Western blot protocols for At1g78820 antibody, researchers should begin with careful sample preparation from Arabidopsis thaliana tissues. Total protein extraction should be performed using a buffer compatible with plant tissues, containing protease inhibitors to prevent degradation of the target protein. For optimal results, use 20-40 μg of total protein per lane and separate by SDS-PAGE using a gel percentage appropriate for the expected molecular weight of the target protein.

After electrophoresis, transfer proteins to a PVDF or nitrocellulose membrane using standard transfer conditions. Block the membrane with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. For primary antibody incubation, prepare a 1:1000 to 1:2000 dilution of the At1g78820 antibody in blocking buffer, and incubate overnight at 4°C with gentle shaking. The exact dilution should be determined empirically for each new lot of antibody.

Following primary antibody incubation, wash the membrane thoroughly with TBST (at least 3 × 10 minutes), then incubate with an appropriate HRP-conjugated secondary antibody (anti-rabbit IgG) at a 1:5000 to 1:10000 dilution for 1 hour at room temperature. After washing again with TBST, visualize the signal using enhanced chemiluminescence detection. Include positive controls (known Arabidopsis tissues expressing At1g78820) and negative controls in each experiment to validate specificity .

What sample preparation methods are recommended for ELISA using the At1g78820 antibody?

For ELISA applications using the At1g78820 antibody, begin with careful extraction of proteins from Arabidopsis thaliana tissues. Fresh or frozen tissue samples should be homogenized in an appropriate extraction buffer (typically PBS with 0.1% Triton X-100 and protease inhibitors) using mechanical disruption methods such as grinding with a mortar and pestle under liquid nitrogen, followed by sonication. After homogenization, centrifuge the samples at 12,000-15,000 g for 15 minutes at 4°C to remove cellular debris.

For indirect ELISA, coat high-binding 96-well plates with purified antigen or protein extract diluted in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. For sandwich ELISA, coat plates with a capture antibody specific to another epitope of the At1g78820 protein. Block non-specific binding sites with 1-5% BSA or non-fat dry milk in PBS for 1-2 hours at room temperature.

Prepare a dilution series of the At1g78820 antibody (typically starting at 1:500 and performing 2-fold serial dilutions) in antibody diluent (usually blocking buffer with 0.05% Tween-20). Incubate for 1-2 hours at room temperature, followed by thorough washing with PBST. Apply an appropriate HRP-conjugated secondary antibody and develop using a suitable substrate. Include positive and negative controls, and generate a standard curve using recombinant At1g78820 protein if quantitative results are needed .

How does At1g78820 expression change during unfolded protein response in Arabidopsis?

The expression of At1g78820 significantly changes during the unfolded protein response (UPR) in Arabidopsis thaliana, as demonstrated in studies using ER stress-inducing agents. When treated with tunicamycin and DTT, At1g78820 shows differential expression patterns that align with other UPR-related genes. Based on comprehensive microarray analysis using Affymetrix GeneChips, At1g78820 exhibits a temporal expression pattern similar to known UPR control genes including BiP, PDI (At1g21750), calreticulin2 (At1g09210), and calnexin1 (At5g61790).

Specific expression fold changes observed after treatment with tunicamycin and DTT are presented in the following table:

This expression pattern suggests that At1g78820 functions as part of the cellular machinery that responds to ER stress. The protein likely contributes to one or more of the three primary UPR functions: improving protein folding and transport, degrading unwanted proteins, or regulating the entry of secretory proteins into the ER. The correlation coefficient of At1g78820 expression with established UPR control genes exceeds 0.95, confirming its role as a UPR-responsive gene. This expression pattern is consistent with the hypothesis that At1g78820 plays a role in maintaining ER homeostasis during cellular stress conditions .

What are the potential cross-reactivity concerns when using At1g78820 antibody in different plant species?

When considering cross-reactivity of the At1g78820 antibody in different plant species, researchers must carefully evaluate sequence homology and epitope conservation. The antibody was raised against a recombinant Arabidopsis thaliana At1g78820 protein and has been specifically validated for reactivity with A. thaliana. Cross-reactivity with other plant species depends primarily on the conservation of the epitope sequence recognized by the antibody.

To assess potential cross-reactivity:

• Perform sequence alignment analysis of the At1g78820 protein sequence with homologous proteins in target species to identify conservation levels.

• Conduct preliminary Western blot experiments with positive controls (A. thaliana samples) alongside samples from the species of interest.

• Include appropriate negative controls and validate results with alternative detection methods.

If cross-reactivity testing is required, researchers should perform serial dilution tests with protein extracts from multiple plant species to determine specificity thresholds. False positive results may occur due to non-specific binding to proteins with similar epitopes, while false negatives may result from species-specific post-translational modifications that interfere with antibody recognition. When working with non-validated species, additional validation steps such as immunoprecipitation followed by mass spectrometry may be necessary to confirm specificity .

How can researchers validate the specificity of At1g78820 antibody in knockout or knockdown experiments?

Validating the specificity of the At1g78820 antibody in knockout or knockdown experiments requires a multi-faceted approach to ensure reliable results. First, researchers should generate or obtain Arabidopsis thaliana lines with At1g78820 gene knockout (via T-DNA insertion, CRISPR-Cas9 editing) or knockdown (via RNAi or artificial microRNA approaches). Then, follow these methodological steps:

• Extract total protein from wild-type plants (positive control), knockout/knockdown plants, and heterozygous plants if available, using identical extraction conditions.

• Perform Western blot analysis using optimized conditions for the At1g78820 antibody. A truly specific antibody should show:

  • Strong signal at the expected molecular weight in wild-type samples

  • Absent signal in knockout samples

  • Reduced signal intensity in knockdown samples proportional to the reduction in transcript levels

• Confirm knockout/knockdown efficiency at the mRNA level using RT-qPCR with primers specific to At1g78820.

• Use densitometric analysis to quantify protein level differences between samples and correlate these with transcript abundance measurements.

• If applicable, perform complementation experiments by expressing the At1g78820 gene in knockout backgrounds, which should restore the antibody signal.

• For advanced validation, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody specifically pulls down At1g78820 protein.

The following table summarizes expected results in different genetic backgrounds:

This comprehensive validation ensures that any experimental observations can be confidently attributed to the specific recognition of At1g78820 protein by the antibody .

What are the recommended approaches for investigating At1g78820 protein interactions in the context of ER stress response?

When investigating At1g78820 protein interactions in the context of ER stress response, researchers should employ multiple complementary approaches to build a comprehensive understanding of interaction networks. Based on the protein's involvement in unfolded protein response pathways, the following methodological strategy is recommended:

• Co-immunoprecipitation (Co-IP) using At1g78820 antibody:

  • Perform under both normal and ER stress conditions (induced by tunicamycin or DTT treatment)

  • Use crosslinking agents to capture transient interactions

  • Identify interaction partners by mass spectrometry

  • Validate key interactions with reciprocal Co-IP experiments

• Yeast two-hybrid (Y2H) screening:

  • Create bait constructs with full-length At1g78820 and domain-specific fragments

  • Screen against Arabidopsis cDNA libraries derived from stressed and unstressed tissues

  • Validate positive interactions with targeted Y2H assays and alternative methods

• Bimolecular Fluorescence Complementation (BiFC):

  • Generate fusion constructs of At1g78820 and candidate interactors with split fluorescent protein fragments

  • Visualize interactions in planta through restored fluorescence

  • Perform under both normal and ER stress conditions to identify stress-dependent interactions

• Proximity-dependent biotin identification (BioID):

  • Create fusion proteins of At1g78820 with a promiscuous biotin ligase

  • Identify proximal proteins through streptavidin purification and mass spectrometry

  • Compare interactome maps under normal vs. stressed conditions

• Genetic interaction studies:

  • Generate double mutants between At1g78820 and candidate interactor genes

  • Analyze synthetic phenotypes under ER stress conditions

  • Quantify UPR marker gene expression in single and double mutants

Particularly relevant protein families to investigate as potential interactors include chaperones (BiP, calreticulins, calnexins), components of the ERAD machinery, and proteins involved in vesicle trafficking that show similar expression patterns during UPR, as identified in comprehensive gene expression studies of tunicamycin and DTT treatments .

How can researchers optimize immunohistochemistry protocols for subcellular localization studies of At1g78820 protein?

For optimal immunohistochemistry (IHC) protocols to determine subcellular localization of At1g78820 protein in Arabidopsis tissues, researchers should implement a carefully designed methodology that preserves cellular architecture while enabling antibody penetration and specific binding. The following comprehensive protocol is recommended:

• Tissue Fixation and Processing:

  • Harvest fresh Arabidopsis tissues and immediately fix in 4% paraformaldehyde in PBS (pH 7.4) for 12-16 hours at 4°C

  • For improved organelle preservation, include 0.1-0.5% glutaraldehyde in the fixative

  • After fixation, dehydrate tissues through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Infiltrate with a paraffin or resin embedding medium suitable for plant tissues

• Sectioning:

  • Prepare thin sections (4-8 μm for light microscopy, 70-100 nm for electron microscopy)

  • Mount on adhesive slides (poly-L-lysine or APTES-coated)

  • Dry overnight at 37°C to ensure adherence

• Antigen Retrieval:

  • Deparaffinize sections in xylene and rehydrate through ethanol series

  • Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) at 95°C for 20-30 minutes

  • Allow to cool slowly to room temperature

• Blocking and Permeabilization:

  • Block with 5% normal serum (from the species of secondary antibody) in PBS containing 0.1-0.3% Triton X-100 for 1-2 hours

  • Include 1% BSA to reduce non-specific binding

• Primary Antibody Incubation:

  • Dilute At1g78820 antibody 1:100 to 1:500 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Optimize dilution through preliminary titration experiments

• Secondary Antibody and Detection:

  • Wash thoroughly with PBS (3 × 10 minutes)

  • Apply fluorescently-labeled anti-rabbit secondary antibody (1:200 to 1:500) for 1-2 hours at room temperature

  • For co-localization studies, include established organelle markers (e.g., ER, Golgi, vacuole)

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1 μg/mL)

  • Mount in anti-fade medium to preserve fluorescence

• Controls and Validation:

  • Include tissue from At1g78820 knockout plants as negative controls

  • Perform peptide competition assays to confirm specificity

  • Compare localization patterns with GFP-tagged At1g78820 in transgenic plants

This protocol should be optimized for each tissue type, as fixation and permeabilization requirements may vary. For high-resolution subcellular localization, consider using super-resolution microscopy techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy .

What techniques can be used to study the effect of post-translational modifications on At1g78820 protein function?

To comprehensively study post-translational modifications (PTMs) of the At1g78820 protein and their functional impact, researchers should employ a multi-technique approach that identifies modifications and elucidates their biological significance. The following methodological workflow is recommended:

• Identification of PTMs:

  • Immunoprecipitate At1g78820 protein using the specific antibody

  • Perform mass spectrometry analysis (LC-MS/MS) with fragmentation methods optimized for PTM detection

  • Use enrichment strategies for specific modifications:

    • Phosphopeptide enrichment using TiO₂ or IMAC

    • Glycopeptide enrichment using lectin affinity chromatography

    • Ubiquitination detection using K-ε-GG antibodies

• Site-directed mutagenesis:

  • Generate point mutations at identified PTM sites (e.g., phospho-mimetic S→D or phospho-deficient S→A mutations)

  • Create transgenic Arabidopsis lines expressing these mutant variants

  • Compare phenotypes and protein function between wild-type and mutant lines

• PTM-specific antibodies:

  • Develop or obtain antibodies specific to identified PTMs (e.g., phospho-specific antibodies)

  • Use these for Western blotting to monitor modification status under different conditions

  • Apply in immunohistochemistry to determine spatial distribution of modified proteins

• In vitro enzymatic assays:

  • Express and purify recombinant At1g78820 protein

  • Perform in vitro modification with relevant enzymes (kinases, glycosyltransferases, etc.)

  • Assess functional consequences on protein activity, stability, or interactions

• PTM dynamics during ER stress:

  • Monitor changes in modification patterns during UPR activation

  • Treatment time course with tunicamycin or DTT (0, 2, 5, 12, 24 hours)

  • Quantify relative abundance of modified vs. unmodified protein forms

The following table outlines potential PTMs and their functional implications for At1g78820:

Understanding these modifications is crucial as they likely serve as regulatory mechanisms that fine-tune At1g78820 function in response to cellular stress conditions, particularly during the unfolded protein response .

How can researchers design experiments to study the role of At1g78820 in different developmental stages and stress conditions?

To comprehensively investigate the role of At1g78820 across developmental stages and stress conditions, researchers should implement a systematic experimental design that integrates genetic, molecular, and physiological approaches. The following methodological framework is recommended:

• Temporal expression analysis:

  • Perform RT-qPCR to quantify At1g78820 transcript levels across developmental stages (seed, seedling, vegetative growth, flowering, senescence)

  • Use the At1g78820 antibody for Western blot analysis to correlate transcript and protein abundance

  • Create promoter-reporter constructs (e.g., pAt1g78820::GUS) to visualize tissue-specific expression patterns

• Genetic resources development:

  • Generate multiple genetic tools:

    • Knockout mutants (T-DNA insertion lines or CRISPR-Cas9 edited)

    • Knockdown lines (RNAi or amiRNA)

    • Overexpression lines (constitutive and inducible)

    • Complementation lines expressing At1g78820 under native promoter in knockout background

    • Fluorescent protein fusions for localization studies

• Stress treatment experimental design:

  • Apply a matrix of stress conditions:

• Multi-omics integration:

  • Perform transcriptome analysis (RNA-seq) comparing wild-type and At1g78820 mutants under control and stress conditions

  • Conduct proteome analysis to identify differentially abundant proteins

  • Analyze metabolome changes to identify affected metabolic pathways

  • Integrate datasets to build comprehensive models of At1g78820 function

• Physiological and phenotypic assessment:

  • Evaluate growth parameters (root length, biomass, leaf area)

  • Measure stress-related physiological markers (electrolyte leakage, lipid peroxidation, ROS accumulation)

  • Assess UPR-specific markers (BiP induction, splicing of bZIP60)

  • Quantify ER stress tolerance through survival rates and recovery assessment

• Protein interaction dynamics:

  • Perform temporally-resolved interaction studies using techniques detailed in FAQ 2.4

  • Compare interactome composition across developmental stages and stress conditions

  • Identify developmental stage-specific or stress-specific interaction partners

This comprehensive experimental design allows researchers to systematically characterize the function of At1g78820 throughout plant development and under various stress conditions, establishing its role in the unfolded protein response and potentially revealing additional functions in plant stress adaptation .

What statistical approaches are most appropriate for analyzing Western blot data when quantifying At1g78820 protein levels?

When quantifying At1g78820 protein levels using Western blot data, researchers should employ robust statistical approaches to ensure reliable and reproducible results. The following comprehensive methodology is recommended:

• Experimental design considerations:

  • Perform a minimum of three biological replicates (independent plant samples)

  • Include two to three technical replicates per biological replicate

  • Incorporate appropriate positive and negative controls (e.g., recombinant protein, knockout samples)

  • Use a loading control (housekeeping protein like actin, tubulin, or GAPDH) for normalization

• Densitometric analysis:

  • Use specialized software (ImageJ, Image Lab, etc.) for densitometric quantification

  • Define regions of interest (ROIs) consistently across all samples

  • Subtract background signal using either local or global background correction

  • Normalize target protein band intensity to loading control within each lane

• Data processing and transformation:

  • Calculate relative protein expression as: (Target protein signal / Loading control signal)

  • Consider log transformation if data shows skewness

  • Test for normal distribution using Shapiro-Wilk or Kolmogorov-Smirnov tests

  • Identify and handle outliers using standardized methods (e.g., Grubbs' test, 1.5×IQR rule)

• Statistical analysis based on experimental comparisons:

  • For two-group comparisons: Use Student's t-test (parametric) or Mann-Whitney U test (non-parametric)

  • For multiple group comparisons: Use one-way ANOVA followed by post-hoc tests (Tukey's HSD, Bonferroni, or Dunnett's test)

  • For factorial designs (e.g., genotype × treatment): Use two-way ANOVA with appropriate post-hoc analysis

  • For time-course experiments: Use repeated measures ANOVA or mixed-effects models

• Statistical reporting:

  • Report mean or median values with appropriate measures of dispersion (standard deviation, standard error, or confidence intervals)

  • Include exact p-values and effect sizes

  • Clearly state the statistical tests used and software version

The following table outlines recommended statistical approaches for different experimental scenarios:

For more complex experimental designs, consider consulting with a biostatistician to ensure appropriate analysis methods are employed. Power analysis should be conducted before experiments to determine adequate sample sizes for detecting biologically meaningful differences in At1g78820 protein levels .

What are the critical considerations for multiplexing At1g78820 antibody with other antibodies in co-localization studies?

When multiplexing the At1g78820 antibody with other antibodies for co-localization studies, researchers must address several critical technical considerations to ensure accurate and interpretable results. The following methodological guidelines will optimize multiplexed immunodetection:

• Antibody selection and compatibility:

  • Primary antibody species considerations:

    • Select primary antibodies raised in different host species (e.g., At1g78820 antibody from rabbit paired with mouse-derived antibodies)

    • If antibodies from the same species are unavoidable, use directly conjugated primary antibodies or sequential immunostaining protocols

  • Isotype considerations:

    • Use secondary antibodies specific to different isotypes when primaries come from the same species

    • Consider using fragment antibodies (Fab) to prevent cross-reactivity

• Spectral considerations:

  • Choose fluorophores with minimal spectral overlap:

    • Select fluorophores with separation of ≥50 nm between emission maxima

    • Consider using quantum dots for narrow emission spectra

  • Account for cellular autofluorescence:

    • Measure autofluorescence in unstained samples

    • Choose fluorophores outside the autofluorescence spectrum of plant tissues

    • Use spectral unmixing for separating overlapping signals

• Optimization of multiplexed protocol:

  • Titrate each antibody individually before multiplexing:

    • Determine optimal concentration for specific signal with minimal background

    • Create a titration matrix to identify optimal concentrations in combination

  • Test for cross-reactivity:

    • Perform controls with each primary and all secondary antibodies

    • Include controls omitting each primary antibody individually

• Sequential staining approach:

  • For challenging combinations, use sequential rather than simultaneous staining:

    • Complete first immunostaining procedure with one primary-secondary pair

    • Block remaining primary antibody binding sites

    • Proceed with second immunostaining procedure

• Imaging and analysis considerations:

  • Microscope settings optimization:

    • Capture single-stained controls to establish imaging parameters

    • Use identical acquisition settings for comparative analysis

  • Quantitative co-localization analysis:

    • Apply appropriate co-localization coefficients (Pearson's, Manders', etc.)

    • Use object-based approaches for discrete structures

    • Employ specialized software for unbiased quantification

The following table outlines potential organelle markers for co-localization with At1g78820:

When reporting results, include representative images of both merged channels and individual channels, alongside quantitative co-localization metrics with appropriate statistical analysis .

How should researchers approach epitope mapping to understand the binding specificity of the At1g78820 antibody?

To comprehensively map the epitope recognized by the At1g78820 antibody and understand its binding specificity, researchers should implement a systematic multi-technique approach. The following methodological workflow is recommended:

• In silico epitope prediction:

  • Begin with computational analysis of the At1g78820 protein sequence to identify potential antigenic regions

  • Use multiple prediction algorithms (Kolaskar & Tongaonkar, Emini Surface Accessibility, Parker Hydrophilicity, etc.)

  • Identify regions with high surface probability, hydrophilicity, and predicted antigenicity

  • Compare with the immunogen sequence used to generate the antibody (recombinant Arabidopsis thaliana At1g78820 protein)

• Peptide array analysis:

  • Design an overlapping peptide library covering the entire At1g78820 sequence

    • Peptides of 12-15 amino acids with 8-10 amino acid overlaps

    • Include alanine scanning for potential epitope regions

  • Synthesize peptides on a membrane or microarray format

  • Probe with At1g78820 antibody and detect binding signal

  • Identify peptides that show strong antibody binding

• Deletion and point mutation analysis:

  • Generate truncated versions of At1g78820 protein

  • Introduce site-directed mutations at residues suspected to be critical for antibody binding

  • Express recombinant proteins and assess antibody binding by Western blot

  • Determine the minimal sequence required for recognition

• Mass spectrometry epitope mapping:

  • Perform hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Compare exchange patterns between free protein and antibody-bound protein

  • Identify regions with reduced exchange rates when antibody is bound

  • Alternatively, use limited proteolysis followed by MS to identify protected regions

• X-ray crystallography or Cryo-EM (advanced):

  • For definitive epitope mapping, obtain crystal structure of antibody Fab fragment complexed with At1g78820 protein or peptide

  • Analyze atomic interactions between antibody and antigen

The following table summarizes the expected outcomes from different epitope mapping techniques:

Understanding the specific epitope recognized by the At1g78820 antibody is crucial for:

• Predicting potential cross-reactivity with homologous proteins

• Assessing whether post-translational modifications might affect antibody binding

• Determining if the epitope remains accessible in different experimental conditions

• Explaining variations in antibody performance across different applications

This information will guide researchers in optimizing experimental protocols and correctly interpreting results when using the At1g78820 antibody in various research applications .