At1g78850 Antibody

What is At1g78850 and what is its biological function in Arabidopsis thaliana?

At1g78850 is a gene in Arabidopsis thaliana (Mouse-ear cress) that encodes a specific protein with UniProt accession number Q9ZVA4 . Based on genomic analysis data, this gene appears to be of interest in plant molecular biology research, particularly in studies related to transcription regulation. The gene has been identified in genomic binding site analyses of the transcription factor HY5, suggesting it may be involved in light-responsive pathways in plants . Comprehensive functional characterization studies should be conducted to fully elucidate its biological role, potentially through gene knockout or overexpression experiments similar to those described for other plant proteins in transcriptome studies .

What are the key specifications of commercially available At1g78850 antibodies?

Commercially available At1g78850 antibodies are typically polyclonal antibodies raised in rabbits against recombinant Arabidopsis thaliana At1g78850 protein . The antibodies are generally supplied in liquid form, preserved in a buffer containing 0.03% Proclin 300 and 50% glycerol in 0.01M PBS at pH 7.4 . These antibodies are purified using antigen affinity methods and are recommended for applications including ELISA and Western blotting . Researchers should note that these are made-to-order reagents with lead times of approximately 14-16 weeks, requiring advance planning for experimental timelines . Upon receipt, proper storage at -20°C or -80°C is critical, with repeated freeze-thaw cycles to be avoided to maintain antibody integrity .

How do researchers validate that an At1g78850 antibody is detecting the correct target?

Validation of At1g78850 antibody should follow a multi-step approach to ensure specificity, selectivity, and reproducibility. The first validation step typically involves Western blotting to determine antibody specificity, confirming a single band at the expected molecular weight of the target protein . For more stringent validation, researchers should use appropriate controls, including:

• Positive controls: Cell lines or plant tissues known to express At1g78850

• Negative controls: Tissue from knockout plants lacking At1g78850 expression

• Overexpression systems: Plants or cells engineered to overexpress At1g78850

If knockout plants are unavailable, alternative approaches include using RNA interference to reduce expression levels or comparing antibody staining patterns with mRNA expression data from other techniques. Cross-validation with a second antibody targeting a different epitope of the same protein can provide additional confidence in specificity .

What are the optimal conditions for using At1g78850 antibody in Western blotting applications?

For optimal Western blotting with At1g78850 antibody, researchers should consider the following methodological approach:

• Sample preparation: Extract total protein from Arabidopsis tissues using a buffer containing appropriate protease inhibitors

• Protein separation: Use 10-12% SDS-PAGE gels with 20-50 μg of total protein per lane

• Transfer: Transfer proteins to PVDF or nitrocellulose membranes at 100V for 60-90 minutes

• Blocking: Block membranes with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute At1g78850 antibody (typically 1:1000 to 1:2000) in blocking buffer and incubate overnight at 4°C

• Washing: Wash membranes 3-4 times with TBST, 5-10 minutes each

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit IgG (1:5000-1:10000) for 1 hour at room temperature

• Detection: Use enhanced chemiluminescence (ECL) detection systems

Always run appropriate controls, including a loading control (like actin or tubulin) and, when possible, samples from At1g78850 knockout or overexpression lines to confirm specificity.

How can At1g78850 antibody be effectively used in immunohistochemistry (IHC) or immunofluorescence (IF) studies?

When using At1g78850 antibody for IHC or IF applications, researchers should follow these methodological guidelines:

• Tissue fixation: Fix plant tissues in 4% paraformaldehyde, considering that fixation time and method significantly affect tissue antigenicity

• Sectioning: Prepare thin sections (5-10 μm) of paraffin-embedded or frozen tissue

• Antigen retrieval: Perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

• Blocking: Block with 5-10% normal serum in PBS with 0.1-0.3% Triton X-100 for 1-2 hours

• Primary antibody: Apply diluted At1g78850 antibody (1:100-1:500) and incubate overnight at 4°C

• Washing: Wash thoroughly with PBS

• Detection: For IF, use fluorophore-conjugated secondary antibodies; for IHC, use HRP-conjugated secondary antibodies and chromogenic substrates

• Controls: Always include no-primary antibody controls and, if possible, tissues from knockout plants

Researchers should note that standardization can be challenging due to numerous pre-analytical, analytical, and post-analytical factors that influence staining in fixed tissues .

What approaches are recommended for quantitative analysis of At1g78850 protein levels?

For quantitative analysis of At1g78850 protein levels, researchers should consider:

• Western blot quantification:

  • Use housekeeping proteins (actin, GAPDH) as loading controls

  • Employ digital image analysis software to measure band intensities

  • Create standard curves using purified recombinant protein when possible

• Quantitative immunofluorescence (QIF):

  • Use consistent image acquisition parameters

  • Include reference standards in each experiment

  • Apply appropriate background subtraction methods

  • Analyze images using specialized software that can quantify fluorescence intensity

• ELISA-based quantification:

  • Develop a sandwich ELISA using At1g78850 antibody paired with another antibody recognizing a different epitope

  • Include a standard curve using recombinant At1g78850 protein

  • Ensure replicate measurements and statistical validation

For all quantitative analyses, researchers should demonstrate reproducibility by showing similar results across multiple independent experiments and antibody lots .

How can researchers study protein-protein interactions involving At1g78850?

To investigate protein-protein interactions involving At1g78850, researchers can employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use At1g78850 antibody to immunoprecipitate the protein complex from plant extracts

  • Analyze co-precipitated proteins by mass spectrometry or Western blotting

  • Validate interactions with reciprocal Co-IP experiments

• Proximity ligation assay (PLA):

  • Apply primary antibodies against At1g78850 and its potential interaction partner

  • Use species-specific PLA probes followed by ligation and amplification

  • Visualize interaction as distinct fluorescent spots where proteins are in close proximity (<40 nm)

• Bimolecular fluorescence complementation (BiFC):

  • Generate fusion constructs of At1g78850 and candidate interactors with split fluorescent protein fragments

  • Express constructs in plant cells and observe for fluorescence reconstitution

  • Include appropriate controls to rule out spontaneous complementation

These methodologies should be used in combination to provide strong evidence for physiologically relevant protein-protein interactions involving At1g78850 .

How can researchers investigate the role of At1g78850 in transcriptional regulation networks?

Based on its inclusion in transcription factor studies, At1g78850 may be involved in transcriptional regulation . To investigate this role, researchers should consider:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Use At1g78850 antibody to immunoprecipitate protein-DNA complexes

  • Identify bound DNA sequences through sequencing (ChIP-seq) or PCR (ChIP-PCR)

  • Compare binding patterns to those of known transcription factors like HY5

• Gene expression analysis:

  • Create and characterize At1g78850 knockout and overexpression lines

  • Perform transcriptome analysis (RNA-seq or microarrays) to identify differentially expressed genes

  • Search for common regulatory elements in affected gene promoters

• Reporter gene assays:

  • Clone candidate target promoters upstream of reporter genes

  • Co-express with At1g78850 to assess transcriptional activation or repression

  • Mutate putative binding sites to confirm direct regulation

These approaches would help position At1g78850 within transcriptional networks, similar to studies conducted for ERF transcription factors described in the literature .

What are appropriate experimental designs for studying At1g78850 in stress response pathways?

To study At1g78850's potential role in stress response pathways, researchers should implement the following experimental approaches:

• Expression profiling under stress conditions:

  • Expose plants to various stressors (oxidative stress, drought, cold, pathogen challenge)

  • Analyze At1g78850 protein levels using validated antibody

  • Compare with transcript levels to identify post-transcriptional regulation

• Genetic approaches:

  • Characterize phenotypes of At1g78850 knockout/overexpression lines under stress

  • Perform complementation studies to confirm phenotype attribution

  • Create double mutants with known stress response genes to identify genetic interactions

• Subcellular localization studies:

  • Use fractionation followed by immunoblotting to track protein location changes during stress

  • Employ immunofluorescence to visualize dynamic relocalization in response to stress

  • Create fluorescent protein fusions to monitor localization in live cells

This experimental framework aligns with approaches used to study stress-responsive proteins in plants, such as the ERF transcription factors and protein kinases mentioned in the research literature .

What are common challenges when working with plant protein antibodies, and how can they be addressed?

Researchers working with plant protein antibodies like At1g78850 antibody frequently encounter these challenges:

• Cross-reactivity issues:

  • Solution: Use extensive validation with knockout controls

  • Perform peptide competition assays to confirm specificity

  • Pre-absorb antibody with plant extracts lacking the target protein

• Low signal strength:

  • Solution: Optimize antigen retrieval methods for fixed tissues

  • Try different antibody concentrations and incubation conditions

  • Consider signal amplification systems (tyramide signal amplification)

• High background:

  • Solution: Increase blocking time and concentration

  • Optimize washing steps (longer, more frequent)

  • Try different blocking agents (BSA, normal serum, commercial blockers)

• Lot-to-lot variability:

  • Solution: Test each new antibody lot against a reference sample

  • Maintain consistent validation protocols

  • Consider creating a large stock of validated antibody

How can researchers resolve contradictory results between antibody detection and transcript analysis for At1g78850?

When antibody-based protein detection results contradict transcript analysis for At1g78850, researchers should:

• Assess post-transcriptional regulation:

  • Measure mRNA stability using actinomycin D chase experiments

  • Investigate potential microRNA-mediated regulation

  • Examine alternative splicing possibilities using RT-PCR with isoform-specific primers

• Evaluate post-translational mechanisms:

  • Check protein stability under different conditions

  • Investigate potential degradation pathways (ubiquitin-proteasome, autophagy)

  • Examine post-translational modifications that might affect antibody recognition

• Validate methodologies:

  • Confirm antibody specificity using knockout controls

  • Verify primer specificity for transcript analysis

  • Use alternative methods for both protein (mass spectrometry) and transcript (RNA-seq) detection

• Consider temporal and spatial factors:

  • Analyze time-course experiments to identify delayed protein expression

  • Examine tissue-specific or subcellular compartment differences

  • Use cell-type specific approaches to resolve potential cellular heterogeneity

This systematic approach addresses the complex relationship between transcription and translation, which often does not follow a simple 1:1 correlation .

What strategies can help improve reproducibility when using At1g78850 antibody across different studies?

To enhance reproducibility when using At1g78850 antibody across different studies, researchers should implement:

• Detailed methodology reporting:

  • Document complete antibody information (vendor, catalog number, lot number)

  • Report all experimental conditions (dilutions, incubation times, buffers)

  • Share validation data in publications and repositories

• Standard operating procedures:

  • Develop and adhere to consistent protocols

  • Use the same positive and negative controls across experiments

  • Standardize image acquisition and analysis parameters

• Cross-validation approaches:

  • Confirm key findings with multiple detection methods

  • Validate with a second independent antibody when possible

  • Compare results with orthogonal techniques (MS-based proteomics)

• Data sharing practices:

  • Deposit complete datasets in appropriate repositories

  • Share detailed antibody validation profiles

  • Report negative results to address publication bias

Implementing these strategies aligns with best practices described in antibody validation literature, which emphasizes the importance of consistency and thorough documentation to improve experimental reproducibility .

What emerging technologies could enhance At1g78850 antibody applications in plant research?

Emerging technologies that could enhance At1g78850 antibody applications include:

• Advanced imaging approaches:

  • Super-resolution microscopy for precise subcellular localization

  • Multiplexed imaging (Imaging Mass Cytometry, CODEX) for simultaneous detection of multiple proteins

  • Live-cell antibody-based imaging using cell-permeable nanobodies

• Single-cell applications:

  • Antibody-based single-cell proteomics

  • In situ protein detection in tissue sections with spatial resolution

  • Microfluidic antibody-based sorting of specific cell populations

• Antibody engineering advances:

  • Recombinant antibody fragments with enhanced tissue penetration

  • Site-specific labeled antibodies for quantitative analyses

  • Bifunctional antibodies for targeted protein degradation studies

• Computational approaches:

  • Machine learning algorithms for antibody staining pattern analysis

  • Prediction tools for epitope accessibility in different experimental conditions

  • Integrated multi-omics data analysis incorporating antibody-derived data

These technological advances would significantly expand the research applications of At1g78850 antibody beyond current capabilities .

How might custom antibody design improve At1g78850 detection specificity and application range?

Custom antibody design could substantially improve At1g78850 detection through:

• Epitope optimization strategies:

  • Targeting highly specific, non-conserved regions of At1g78850

  • Developing antibodies against multiple distinct epitopes

  • Creating antibodies specific to different protein conformations or modifications

• Advanced immunization approaches:

  • Using structured-based immunogen design to enhance specificity

  • Implementing phage display selection against specific epitopes

  • Employing negative selection against closely related proteins

• Recombinant antibody technologies:

  • Developing single-chain variable fragments (scFvs) for improved penetration

  • Creating antibody fusion proteins with reporter enzymes or fluorescent proteins

  • Engineering antibodies with controlled affinity for quantitative applications

• Computational design methods:

  • Using machine learning models to predict antibody specificity

  • Employing energy functions to optimize binding to target epitopes

  • Designing antibodies with customized specificity profiles for discriminating very similar ligands

These approaches align with recent advances in computational antibody design described in the literature, which enable the creation of antibodies with precisely defined binding characteristics .