At1g31072 Antibody

What is At1g31072 protein and why is it studied in Arabidopsis thaliana?

At1g31072 (UniProt No: Q9SA01) is a protein found in Arabidopsis thaliana (Mouse-ear cress), a model organism extensively used in plant biology research. This protein is studied as part of understanding plant molecular biology and cellular processes. The specific antibody against this protein is raised in rabbits using recombinant Arabidopsis thaliana At1g31072 protein as the immunogen . The antibody enables researchers to detect and investigate the protein's expression, localization, and function in various experimental contexts such as stress responses, developmental processes, or plant-pathogen interactions.

What are the validated applications for At1g31072 Antibody?

At1g31072 Antibody has been validated for specific research applications including:

• Enzyme-Linked Immunosorbent Assay (ELISA): For quantitative detection of At1g31072 protein in sample preparations

• Western Blotting (WB): For identification and semi-quantitative analysis of At1g31072 protein in plant tissue extracts

When designing experiments, researchers should consider that antibody performance can vary between applications, necessitating optimization for each specific experimental context. Similar to other research antibodies, validation experiments should be conducted to confirm specificity in your experimental system, as demonstrated with other antibodies in the literature .

What are the optimal storage and handling conditions for At1g31072 Antibody?

To maintain antibody efficacy throughout your research project:

• Store the antibody at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles, which can compromise antibody activity

• The antibody is supplied in liquid form with 0.03% Proclin 300 as a preservative

For long-term projects, consider aliquoting the antibody into single-use volumes to minimize freeze-thaw cycles. This practice is consistent with standard protocols for antibody preservation, regardless of the specific target protein.

How should researchers validate At1g31072 Antibody specificity for reliable results?

Antibody validation is critical for ensuring experimental reproducibility. For At1g31072 Antibody, implement these validation strategies:

• Positive and Negative Controls: Include wild-type Arabidopsis samples alongside knockout/knockdown lines if available

• Blocking Peptide Assay: Pre-incubate the antibody with excess recombinant At1g31072 protein to confirm specificity

• Secondary Antibody Controls: Perform parallel experiments with secondary antibody only to assess background

• Cross-Reactivity Assessment: Test the antibody on related plant species to determine specificity

This validation approach mirrors techniques used for other research antibodies, such as the AT1 receptor antibody described in result , where antibody specificity was confirmed using transfected cells expressing the target protein.

What are the considerations for optimizing Western blot protocols with At1g31072 Antibody?

Achieving optimal Western blot results requires attention to several parameters:

Much like the approaches used in antibody analysis described in search result , optimization should include careful consideration of experimental conditions to enhance specificity and reduce background signals.

How can researchers troubleshoot non-specific binding issues with At1g31072 Antibody?

When encountering non-specific binding, implement these systematic troubleshooting approaches:

• Increase Washing Stringency: Extend TBST washing steps to 15 minutes with 3-5 changes of buffer

• Optimize Blocking: Test different blocking agents (milk, BSA, or commercial blockers)

• Adjust Antibody Concentration: Further dilute the primary antibody if background is excessive

• Add Detergents: Include 0.1-0.3% Triton X-100 in antibody diluent to reduce hydrophobic interactions

• Pre-absorb Antibody: Incubate diluted antibody with plant material from non-target species

These approaches are consistent with standard immunological techniques used to improve antibody specificity, similar to the optimization procedures employed in developing therapeutic antibodies described in result .

What is the recommended protocol for immunoprecipitation using At1g31072 Antibody?

For successful immunoprecipitation of At1g31072 protein from Arabidopsis samples:

• Sample Preparation:

  • Grind 100-200 mg plant tissue in liquid nitrogen

  • Add 500 μl ice-cold IP lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, protease inhibitor cocktail)

  • Incubate with rotation for 30 minutes at 4°C

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

• Pre-clearing:

  • Incubate supernatant with 20 μl Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Add 2-5 μg of At1g31072 Antibody to pre-cleared lysate

  • Incubate overnight at 4°C with gentle rotation

  • Add 30 μl Protein A/G beads, incubate for 2 hours at 4°C

  • Collect beads by centrifugation, wash 4 times with wash buffer

• Elution and Analysis:

  • Elute with SDS sample buffer at 95°C for 5 minutes

  • Analyze by SDS-PAGE and Western blotting

This protocol follows principles similar to those used for other research antibodies and can be adapted based on the specific binding characteristics of the At1g31072 Antibody.

How can mass spectrometry complement At1g31072 Antibody-based research?

Mass spectrometry provides powerful validation and extension of antibody-based findings:

• Confirming Antibody Specificity:

  • Immunoprecipitate using At1g31072 Antibody

  • Separate proteins by SDS-PAGE

  • Excise bands of interest

  • Perform tryptic digestion and LC-MS/MS analysis

  • Compare peptide sequences with At1g31072 protein sequence

• Identifying Interaction Partners:

  • Perform co-immunoprecipitation with At1g31072 Antibody

  • Analyze entire precipitated fraction by LC-MS/MS

  • Use protein databases to identify co-precipitated proteins

• Protocol Integration:

  • Use antibody for initial purification

  • Apply MS protocols similar to those described in search result

  • Consider HPLC separation combined with TOF MS analysis for accurate mass determination

This approach leverages the specificity of the antibody with the analytical power of mass spectrometry for comprehensive protein characterization.

What controls should be included in immunofluorescence studies with At1g31072 Antibody?

For rigorous immunofluorescence experiments, incorporate these essential controls:

• Specificity Controls:

  • Primary antibody omission

  • Isotype control (rabbit IgG at equivalent concentration)

  • Blocking peptide competition

  • Genetically modified plants lacking the target protein

• Technical Controls:

  • Autofluorescence control (sample without any antibody)

  • Secondary antibody only

  • Known marker proteins for co-localization studies

• Sample Processing Controls:

  • Fixation time series (2% paraformaldehyde for 15, 30, 60 minutes)

  • Permeabilization optimization (0.1%, 0.3%, 0.5% Triton X-100)

  • Antigen retrieval comparison if applicable

These controls are essential for validating the specificity of immunostaining patterns and follow principles similar to those employed in the specificity testing of the monoclonal antibodies described in search result .

How can At1g31072 Antibody be used in protein-protein interaction studies?

The antibody can be deployed in multiple complementary approaches to study protein interactions:

• Co-Immunoprecipitation (Co-IP):

  • Use At1g31072 Antibody to precipitate the target protein

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

  • Compare results under different plant growth conditions

• Proximity Ligation Assay (PLA):

  • Combine At1g31072 Antibody with antibodies against suspected interaction partners

  • Apply oligonucleotide-conjugated secondary antibodies

  • Detect amplification signal only when proteins are in close proximity (<40 nm)

• Förster Resonance Energy Transfer (FRET):

  • Label At1g31072 Antibody with donor fluorophore

  • Label partner protein antibody with acceptor fluorophore

  • Measure energy transfer as evidence of protein proximity

These methodologies extend beyond basic detection to provide insight into protein function and interaction networks, similar to approaches used in studying receptor interactions described in search result .

What are the considerations for adapting At1g31072 Antibody protocols for use with different plant species?

When extending research to species beyond Arabidopsis thaliana:

• Sequence Homology Assessment:

  • Align At1g31072 sequences across target species

  • Focus on conservation within the antibody epitope region

  • Predict cross-reactivity based on amino acid similarity

• Protocol Modifications:

  • Adjust protein extraction buffers based on species-specific tissue composition

  • Optimize antibody concentration (typically higher for cross-species applications)

  • Consider longer incubation times to accommodate lower-affinity binding

• Validation Requirements:

  • Verify antibody recognition using recombinant proteins from target species

  • Include additional controls when working with non-Arabidopsis species

  • Consider Western blot analysis prior to other applications to confirm binding

This cross-species adaptation approach follows principles similar to those used in developing antibodies with broader species reactivity as mentioned in search result .

How can researchers quantitatively analyze Western blot data generated with At1g31072 Antibody?

For rigorous quantitative analysis:

• Densitometry Workflow:

  • Capture high-resolution, unsaturated digital images

  • Use software such as ImageJ to define regions of interest

  • Subtract background signal from each band

  • Normalize to loading controls (e.g., actin, tubulin, or total protein stain)

• Statistical Considerations:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests based on experimental design

  • Report both means and measures of dispersion (standard deviation or standard error)

• Recommended Controls for Quantitation:

  • Include calibration curves with known quantities of recombinant protein

  • Apply multiple normalization references for robust analysis

  • Consider using fluorescent secondary antibodies for wider linear detection range

This quantitative approach provides more rigorous data interpretation than qualitative assessment alone, similar to the analytical methods applied to antibody characterization in search result .

How can engineered antibody technologies enhance At1g31072 research applications?

Recent advances in antibody engineering offer opportunities to extend the utility of At1g31072 research:

• Fragment-Based Derivatives:

  • Fab or scFv fragments may provide improved tissue penetration in plant samples

  • Smaller antibody fragments could enhance immunoprecipitation efficiency

  • Consider testing commercially available enzymatic fragmentation kits

• Bispecific Adaptations:

  • Following principles like those in the ATG-101 bispecific antibody , design approaches targeting At1g31072 alongside known interaction partners

  • Employ sequential immunoprecipitation strategies to isolate specific protein complexes

• Implementation Considerations:

  • Evaluate cost-benefit of custom antibody development versus commercial offerings

  • Assess the necessity of advanced formats based on specific research questions

  • Consider collaboration with antibody engineering laboratories for specialized applications

These approaches build upon the antibody design principles described in search result , applying them to plant biology research contexts.

What emerging analytical techniques can complement At1g31072 Antibody-based research?

Integrating cutting-edge analytical methods can provide deeper insights:

• High-Resolution Microscopy:

  • Super-resolution techniques (STED, PALM, STORM) for nanoscale localization

  • Expand microscopy for physical enlargement of samples to improve resolution

  • Live-cell imaging with minimally disruptive antibody fragments

• Single-Cell Analysis:

  • Flow cytometry of protoplasts labeled with At1g31072 Antibody

  • Single-cell western blotting for heterogeneity assessment

  • Mass cytometry (CyTOF) for multiplexed protein detection

• Computational Integration:

  • Machine learning algorithms for pattern recognition in localization studies

  • Systems biology approaches to integrate antibody-based data with transcriptomics

  • Protein structure prediction to enhance understanding of antibody-epitope interactions

These advanced techniques extend traditional antibody applications into emerging research territories, providing multi-dimensional data similar to the comprehensive analytical approaches described in search result .