At1g68190 Antibody

What is At1g68190 and why is it significant for plant research?

At1g68190 is a putative zinc finger protein from Arabidopsis thaliana (Mouse-ear cress) that plays potential roles in transcriptional regulation and stress responses. The protein consists of 356 amino acids and contains zinc finger domains that enable DNA binding capabilities . Zinc finger proteins in plants are significant as they regulate crucial developmental processes, stress responses, and various signaling pathways. Research on At1g68190 contributes to our understanding of gene regulation mechanisms in plants and potential applications in crop improvement and stress tolerance.

What detection methods can be used with At1g68190 antibodies?

At1g68190 antibodies can be applied in multiple detection methods, similar to other plant protein antibodies. Based on standard antibody applications, researchers can use these antibodies for:

• Western blotting (WB) for protein expression analysis

• Immunoprecipitation (IP) to study protein interactions

• Immunofluorescence (IF) for cellular localization studies

• Immunohistochemistry (IHC) to study tissue distribution

• Enzyme-linked immunosorbent assay (ELISA) for quantitative protein detection

The selection of detection method depends on the specific research question being addressed. For optimal results, validate each application independently as antibody performance can vary across different detection platforms.

How should At1g68190 antibodies be stored and handled for maximum stability?

For optimal stability and performance of At1g68190 antibodies, adhere to these storage and handling guidelines:

• Store antibodies at -20°C for long-term storage

• For working solutions, aliquot and store at 4°C for up to one week

• Avoid repeated freeze-thaw cycles as this significantly reduces antibody activity

• When handling antibodies, use sterile techniques to prevent contamination

• If conjugated versions are used, protect from light exposure

• Always centrifuge briefly before opening to collect liquid at the bottom of the tube

Proper storage conditions are crucial for maintaining antibody stability and binding capacity over time.

How should At1g68190 antibody specificity be validated before experimental use?

Comprehensive validation of At1g68190 antibody specificity is essential before experimental applications. Implement this validation workflow:

A multi-method validation approach significantly increases confidence in antibody specificity and experimental results. Document validation results thoroughly in your research protocols.

What controls should be included when performing western blot analysis with At1g68190 antibodies?

When conducting western blot analysis with At1g68190 antibodies, include these essential controls:

• Positive control: Wild-type Arabidopsis thaliana tissue sample known to express At1g68190

• Negative control: Tissue from knockout/knockdown plants or tissues where the protein is not expressed

• Loading control: Probe for housekeeping proteins (e.g., actin, tubulin) to ensure equal protein loading

• Blocking peptide control: Pre-incubate antibody with excess target peptide to demonstrate binding specificity

• Secondary antibody-only control: Omit primary antibody to detect non-specific binding of secondary antibody

• Molecular weight ladder: To confirm the target protein appears at the expected size (~40 kDa for At1g68190)

These controls help distinguish specific antibody binding from experimental artifacts and increase data reliability.

How can I optimize immunoprecipitation protocols for At1g68190 antibodies to study protein interactions?

Optimizing immunoprecipitation (IP) with At1g68190 antibodies requires systematic protocol refinement:

• Antibody selection: Choose antibodies validated for IP applications with confirmed specificity against native At1g68190

• Lysis buffer optimization: Test multiple buffer compositions to preserve protein interactions while efficiently extracting At1g68190:

  • RIPA buffer for stronger extraction

  • NP-40 buffer for preserving more labile interactions

  • Add protease inhibitors and phosphatase inhibitors freshly

• Antibody immobilization method:

  • Direct coupling to beads may improve results compared to protein A/G methods

  • Consider using agarose-conjugated antibodies for reduced background

• IP conditions optimization:

  • Test various antibody concentrations (1-10 μg per sample)

  • Optimize incubation times (2 hours vs. overnight)

  • Adjust washing stringency based on interaction strength

• Detection method: Follow with western blotting or mass spectrometry for interaction partner identification

For detecting weak or transient interactions, consider crosslinking approaches before cell lysis to stabilize complexes.

What are the most common reasons for false positive or false negative results when using At1g68190 antibodies, and how can they be addressed?

Understanding and addressing potential sources of error with At1g68190 antibodies is critical for reliable results:

Implementing a systematic troubleshooting approach based on these considerations will significantly improve the reliability of experimental results with At1g68190 antibodies.

How can At1g68190 antibodies be used to study zinc finger protein dynamics during stress responses in plants?

At1g68190 antibodies can be powerful tools for investigating zinc finger protein dynamics during plant stress responses through several advanced approaches:

• Time-course immunoblotting: Monitor At1g68190 protein levels across different stress conditions and time points to establish expression profiles

• Chromatin immunoprecipitation (ChIP) assays: Identify DNA binding sites of At1g68190 during various stress conditions:

  • Use At1g68190 antibodies to pull down protein-DNA complexes

  • Analyze bound DNA sequences by qPCR or sequencing (ChIP-seq)

  • Map binding site changes in response to different stressors

• Co-immunoprecipitation coupled with mass spectrometry: Identify stress-specific interaction partners:

  • Perform IP-MS under various stress conditions

  • Compare protein interaction networks between normal and stress conditions

  • Quantify changes in interaction strength using NSAF values

• Immunolocalization studies: Track subcellular relocalization during stress:

  • Use fluorescent-conjugated secondary antibodies against At1g68190 primary antibodies

  • Apply confocal microscopy to observe protein trafficking between cellular compartments

• Proximity ligation assays: Visualize and quantify protein-protein interactions in situ:

  • Combine At1g68190 antibodies with antibodies against suspected interaction partners

  • Measure interaction dynamics during stress response development

These methodologies collectively provide a comprehensive view of how At1g68190 functions within stress response pathways in plants.

What approaches can be used to assess post-translational modifications of At1g68190 using specific antibodies?

Investigating post-translational modifications (PTMs) of At1g68190 requires specialized antibody-based approaches:

• Modification-specific antibodies: When available, use antibodies that specifically recognize phosphorylated, ubiquitinated, or SUMOylated forms of At1g68190

• Sequential immunoprecipitation:

  • First IP: Use At1g68190 antibodies to capture the protein

  • Second IP: Probe with antibodies against specific modifications (phospho-serine/threonine/tyrosine, ubiquitin, SUMO)

  • Western blot analysis to confirm modifications

• Mass spectrometry of immunoprecipitated protein:

  • Perform IP with At1g68190 antibodies

  • Analyze captured protein by MS to identify modification sites and types

  • Compare PTM profiles under different conditions

• Phosphatase treatment controls:

  • Split samples and treat one with phosphatase before immunoblotting

  • Compare migration patterns to identify phosphorylation-dependent mobility shifts

• In vitro kinase assays with immunoprecipitated protein:

  • Capture At1g68190 with specific antibodies

  • Subject to in vitro kinase reactions with suspected kinases

  • Detect phosphorylation using phospho-specific antibodies or radioactive ATP

These methodologies provide complementary information about At1g68190 PTMs and their functional significance in plant signaling pathways.

How should researchers quantify and normalize At1g68190 expression levels across different experimental conditions?

Accurate quantification and normalization of At1g68190 expression requires rigorous methodological approaches:

• Western blot quantification:

  • Use calibrated standards of recombinant At1g68190 protein for absolute quantification

  • Apply digital image analysis software (ImageJ, Image Lab) for densitometry

  • Establish linear detection range for accurate quantification

  • Always normalize to appropriate loading controls (GAPDH, actin, tubulin)

• ELISA-based quantification:

  • Develop a standard curve using purified recombinant At1g68190 protein

  • Ensure samples fall within the linear range of the standard curve

  • Calculate protein concentration based on 4-parameter logistic regression models

• Normalization strategies across experiments:

  • Use the same amount of total protein in each experiment

  • Apply multiple reference proteins for normalization

  • Consider using total protein normalization methods (Ponceau S, SYPRO Ruby)

  • Report relative fold changes rather than absolute values when comparing across experiments

• Statistical analysis requirements:

  • Perform at least three biological replicates for statistical validity

  • Use appropriate statistical tests (t-test, ANOVA) depending on experimental design

  • Report p-values and confidence intervals for all quantitative comparisons

These approaches ensure reliable quantification of At1g68190 expression levels and facilitate meaningful comparisons across diverse experimental conditions.

How can contradictory results between different antibody-based methods for At1g68190 be resolved?

When facing contradictory results between different antibody-based methods for At1g68190, implement this systematic resolution approach:

• Antibody validation reassessment:

  • Re-validate antibody specificity using multiple methods

  • Test for potential cross-reactivity with related zinc finger proteins

  • Consider using alternative antibodies targeting different epitopes

• Method-specific considerations:

  • Western blot: Denaturing conditions may affect epitope accessibility

  • Immunofluorescence: Fixation methods may alter epitope conformation

  • ELISA: Native protein folding may obscure certain epitopes

  • IP-MS: Protein complexes may mask antibody binding sites

• Orthogonal validation approaches:

  • Complement antibody-based methods with non-antibody techniques

  • Use genetic approaches (knockout/knockdown) to confirm specificity

  • Apply mass spectrometry for unbiased protein identification

  • Implement recombinant tagging strategies for verification

• Systematic troubleshooting protocol:

  Method Combination	Discrepancy Type	Resolution Approach
  WB vs. IF	Detection in IF but not WB	Test multiple extraction buffers; check protein solubility
  WB vs. ELISA	Different quantification results	Verify linear range of both assays; check native vs. denatured conformations
  IP-MS vs. co-IP	Different interaction partners	Evaluate buffer stringency; test mild vs. strong detergents
  IF vs. IHC	Different localization patterns	Compare fixation methods; validate tissue preparation protocols

• Collaboration and independent verification:

  • Have independent researchers replicate critical experiments

  • Consult with antibody production specialists about potential limitations

Reconciling contradictory results often leads to deeper insights into protein biology and improved experimental designs.

What emerging technologies are improving the specificity and applications of plant protein antibodies like those for At1g68190?

Several cutting-edge technologies are enhancing antibody specificity and expanding applications for plant research:

• Recombinant antibody technologies:

  • Single-chain variable fragments (scFv) and nanobodies offer improved specificity

  • Display technologies (phage, yeast) enable selection of high-affinity binders

  • Sequence-defined recombinant antibodies provide consistent reproducibility

• Multiplexed antibody platforms:

  • Suspension bead assays (Luminex) allow simultaneous testing against multiple antigens

  • Microarray-based antibody validation improves specificity assessment

  • Multiplex imaging capabilities enable co-localization studies

• Proximity-based detection methods:

  • Proximity ligation assays offer single-molecule interaction detection

  • FRET-based approaches visualize protein interactions in live cells

  • Split protein complementation assays provide functional interaction data

• Antibody engineering approaches:

  • Structure-guided antibody design improves specificity

  • Antibody conjugation techniques expand detection capabilities

  • Site-specific biotinylation improves orientation in binding assays

• Emerging validation strategies:

  • CRISPR-based knockout controls provide gold-standard validation

  • Machine learning algorithms predict cross-reactivity risks

  • IP-MS standardization improves interaction partner identification

These technological advances will continue to enhance the reliability and expand the applications of At1g68190 antibodies in plant research.

How can researchers integrate antibody-based assays with other molecular techniques to build comprehensive models of At1g68190 function in plant development?

Integrating antibody-based assays with complementary molecular techniques creates a powerful systems biology approach to understanding At1g68190 function:

• Multi-omics integration framework:

  • Combine antibody-based protein abundance/localization data with transcriptomics

  • Correlate ChIP-seq binding data with RNA-seq expression changes

  • Integrate PTM profiles with metabolomics to link modifications to metabolic outcomes

• Genetic and antibody-based approaches synergy:

  • Use CRISPR/Cas9 to generate targeted At1g68190 mutations

  • Apply antibodies to track resulting protein changes

  • Combine with phenotypic analyses to link molecular changes to plant development

• Temporal and spatial dynamics assessment:

  • Time-course antibody studies track protein changes during development

  • Tissue-specific immunolocalization maps expression domains

  • Combine with live cell imaging of fluorescent reporters for dynamic analysis

• Protein complex network mapping:

  • Use antibodies for sequential co-IP followed by mass spectrometry

  • Identify stable and transient interaction partners across development

  • Build interaction networks that change during developmental transitions

• Computational integration and modeling:

  • Develop mathematical models incorporating antibody-derived quantitative data

  • Predict regulatory networks based on ChIP-seq and expression correlations

  • Test model predictions with targeted antibody-based experiments

This integrated approach generates testable hypotheses about At1g68190 function while building comprehensive models of its role in plant development and stress responses.