At1g61060 Antibody

What is At1g61060 and why is it studied in Arabidopsis thaliana?

At1g61060 refers to a specific gene locus in Arabidopsis thaliana (Mouse-ear cress), a model organism widely used in plant molecular biology research. This gene encodes a protein with UniProt accession number Q9C948 . Arabidopsis thaliana serves as an excellent model system due to its small genome, short life cycle, and genetic tractability. The At1g61060 gene product has been studied for its potential roles in plant development, stress responses, and cellular signaling pathways. Researchers typically use At1g61060 antibodies to detect, quantify, and localize the corresponding protein in various experimental contexts.

What applications can At1g61060 Antibody be used for in plant research?

At1g61060 Antibody can be utilized in multiple experimental applications in plant research:

• Western blotting for protein detection and semi-quantitative analysis

• Immunohistochemistry (IHC) for protein localization in plant tissues

• Immunoprecipitation (IP) for protein isolation and interaction studies

• Chromatin immunoprecipitation (ChIP) if the protein has DNA-binding properties

• Enzyme-linked immunosorbent assay (ELISA) for quantitative analysis

Like many research antibodies, At1g61060 Antibody is typically available in formats suitable for these common applications, though specific validation for each method should be performed prior to experimental use .

What are the recommended storage and handling conditions for At1g61060 Antibody?

Based on standard antibody storage protocols similar to other research antibodies, At1g61060 Antibody should be stored at -20°C for long-term preservation. For short-term use within a few weeks, storage at 4°C is typically acceptable . The antibody is commonly supplied in a buffer containing PBS with glycerol and a preservative like thimerosal to maintain stability .

To ensure optimal performance:

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon receipt

• Keep on ice when in use but avoid prolonged exposure

• Centrifuge briefly before opening the tube to collect all liquid at the bottom

• Do not expose to high temperatures or direct sunlight

• Follow manufacturer's specific recommendations for reconstitution if supplied in lyophilized form

Advanced Research Methodologies

For optimal Western blotting results with At1g61060 Antibody, consider the following protocol recommendations:

• Sample preparation:

  • Extract proteins from Arabidopsis tissues using a plant-specific extraction buffer containing protease inhibitors

  • Use approximately 30 μg of total protein per lane based on standard protocols

  • Denature samples in SDS loading buffer at 95°C for 5 minutes

• Gel electrophoresis and transfer:

  • Use 12% SDS-PAGE gels for optimal separation

  • Transfer to PVDF or nitrocellulose membrane at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

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

  • Dilute primary At1g61060 Antibody at 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Wash 3-5 times with TBST, 5 minutes each

  • Incubate with appropriate HRP-conjugated secondary antibody (anti-rabbit) at 1:5000 for 1 hour

  • Wash 3-5 times with TBST

• Detection:

  • Develop using enhanced chemiluminescence (ECL) substrate

  • Expose to X-ray film or image using a digital imaging system

Optimization may be necessary for your specific experimental conditions, including testing different antibody dilutions, incubation times, and blocking reagents.

How can I use At1g61060 Antibody for immunohistochemistry in plant tissues?

Immunohistochemistry (IHC) in plant tissues requires special considerations due to the unique cellular structures. For At1g61060 Antibody IHC applications:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde in PBS for 4-12 hours

  • Dehydrate through an ethanol series and embed in paraffin

  • Section tissues at 5-10 μm thickness and mount on positively charged slides

• Antigen retrieval:

  • Deparaffinize sections with xylene and rehydrate through decreasing ethanol series

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

  • Cool gradually to room temperature

• Staining procedure:

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Block non-specific binding with 5% normal goat serum for 1 hour

  • Apply At1g61060 Antibody at 1:100 dilution and incubate overnight at 4°C

  • Wash 3 times with PBS

  • Apply HRP-conjugated secondary antibody for 1 hour at room temperature

  • Develop with DAB substrate and counterstain with hematoxylin

  • Dehydrate, clear, and mount

• Controls:

  • Include negative controls (omitting primary antibody)

  • Use tissue from knockout/knockdown plants as specificity controls

Autofluorescence can be a significant challenge in plant tissues, so consider using alternative detection methods like enzyme-based systems if fluorescent detection proves problematic.

What approaches can address contradictory results when using At1g61060 Antibody?

When faced with contradictory results using At1g61060 Antibody, employ these troubleshooting strategies:

• Revalidate antibody specificity:

  • Test antibody lot-to-lot variation

  • Perform additional specificity tests including epitope mapping

  • Consider obtaining a different antibody targeting another region of the protein

• Examine experimental variables:

  • Growth conditions of plants (light, temperature, media composition)

  • Developmental stage and tissue type

  • Extraction and sample preparation methods

  • Protein modifications (phosphorylation, glycosylation, etc.)

• Implement alternative detection methods:

  • Combine antibody-based detection with transcript analysis (qRT-PCR)

  • Use mass spectrometry for protein identification

  • Consider reporter gene fusion approaches (GFP, YFP)

• Systematic experimental matrix:
  Create a comprehensive testing matrix like the example below:

• Literature verification:

  • Compare findings with published data on At1g61060 expression and localization

  • Contact corresponding authors of relevant publications for technical advice

How can I optimize immunoprecipitation protocols for studying At1g61060 protein interactions?

For effective immunoprecipitation (IP) of At1g61060 and its interacting partners:

• Extraction buffer optimization:

  • Test different lysis buffers (mild to stringent) to maintain protein interactions

  • Consider native conditions with buffers containing 0.1-0.5% NP-40 or Triton X-100

  • Include protease and phosphatase inhibitors to prevent degradation

• Pre-clearing and antibody binding:

  • Pre-clear lysates with Protein A/G beads to reduce non-specific binding

  • Use 2-5 μg of At1g61060 Antibody per 500 μg of total protein

  • Incubate antibody with lysate overnight at 4°C with gentle rotation

• Bead selection and elution:

  • Use magnetic or agarose Protein A/G beads based on host species (rabbit for At1g61060 Antibody)

  • Wash beads thoroughly (4-5 times) with decreasing salt concentrations

  • Elute bound proteins with SDS sample buffer or low pH buffer

  • For interactome studies, consider mild elution conditions to maintain complex integrity

• Crosslinking considerations:

  • For transient interactions, consider using crosslinking agents (DSP, formaldehyde)

  • Optimize crosslinking time and concentration to capture authentic interactions

• Validation of interactions:

  • Perform reverse IP with antibodies against suspected interacting partners

  • Confirm specificity with appropriate controls (IgG, pre-immune serum)

  • Validate key interactions using alternative methods (Y2H, BiFC)

How does At1g61060 expression change under different environmental stresses?

Understanding At1g61060 expression patterns under various stress conditions requires comprehensive experimental design. While specific data for At1g61060 is limited in the search results, the following approach is recommended for analyzing expression changes:

• Stress treatment experimental design:

  • Subject Arabidopsis plants to common stresses: drought, salt, heat, cold, pathogen infection

  • Collect samples at multiple time points (0, 1, 3, 6, 12, 24, 48 hours)

  • Include proper controls for each stress condition

• Multi-level expression analysis:

  • Transcript analysis: qRT-PCR, RNA-seq for mRNA levels

  • Protein analysis: Western blotting with At1g61060 Antibody for protein levels

  • Post-translational modifications: Phospho-specific antibodies if available

• Tissue-specific expression:

  • Compare expression across different tissues (roots, leaves, stems, flowers)

  • Consider using tissue-specific promoters for detailed analysis

• Visualization of results:
  Create comprehensive expression heat maps like the example template below:

This systematic approach will provide insights into the potential roles of At1g61060 in stress response pathways.

What are the best approaches for studying At1g61060 localization at the subcellular level?

To determine the subcellular localization of At1g61060 protein:

• Immunofluorescence microscopy:

  • Fix Arabidopsis tissues with 4% paraformaldehyde

  • Permeabilize cell walls and membranes appropriately (enzymatic digestion or detergent)

  • Use At1g61060 Antibody as primary and fluorophore-conjugated secondary antibody

  • Co-stain with organelle markers (nucleus, chloroplast, ER, Golgi, mitochondria)

  • Analyze using confocal microscopy

• Biochemical fractionation:

  • Separate cellular components through differential centrifugation

  • Isolate organelles using density gradient centrifugation

  • Detect At1g61060 in fractions using Western blotting

  • Verify fractionation quality with known organelle marker proteins

• Fluorescent protein fusion approaches:

  • Create N- and C-terminal GFP/YFP fusions with At1g61060

  • Express in Arabidopsis using appropriate promoters

  • Visualize in live cells using confocal microscopy

  • Validate functionality of fusion proteins

• Electron microscopy:

  • Use immunogold labeling with At1g61060 Antibody

  • Visualize at ultrastructural level to precisely determine localization

  • Quantify gold particle distribution across cellular compartments

A combination of these approaches provides the most reliable determination of subcellular localization, as each method has its own strengths and limitations.

How can I use At1g61060 Antibody in chromatin immunoprecipitation (ChIP) studies?

If At1g61060 has DNA-binding properties or associates with chromatin, ChIP can be a valuable technique:

• ChIP protocol optimization:

  • Cross-link Arabidopsis tissues with 1% formaldehyde for 10-15 minutes

  • Quench with 125mM glycine

  • Isolate nuclei and sonicate chromatin to 200-500bp fragments

  • Immunoprecipitate with At1g61060 Antibody (4-5μg per reaction)

  • Include appropriate controls (IgG, input)

  • Reverse cross-links and purify DNA

  • Analyze by qPCR or sequencing (ChIP-seq)

• ChIP-seq analysis considerations:

  • Ensure sufficient sequencing depth (>20 million reads)

  • Use appropriate peak calling algorithms (MACS2)

  • Perform motif analysis on binding regions

  • Integrate with transcriptomic data to identify potential target genes

• Validation strategies:

  • Confirm binding to selected regions by ChIP-qPCR

  • Perform electrophoretic mobility shift assays (EMSA)

  • Test functional effects through reporter gene assays

What emerging technologies can enhance research using At1g61060 Antibody?

Several cutting-edge technologies can be integrated with At1g61060 Antibody research:

• Proximity labeling approaches:

  • BioID or TurboID fusion with At1g61060

  • APEX2-based proximity labeling

  • Identification of proximal proteins in native cellular contexts

• Advanced imaging techniques:

  • Super-resolution microscopy (STORM, PALM)

  • Live-cell imaging with lattice light-sheet microscopy

  • Correlative light and electron microscopy (CLEM)

• Proteomics integration:

  • Quantitative TMT/iTRAQ proteomics

  • Parallel reaction monitoring (PRM) for targeted quantification

  • Thermal proteome profiling for identifying binding partners

• Single-cell approaches:

  • Single-cell proteomics

  • Spatial transcriptomics correlated with protein localization

  • Microfluidic applications for single-cell protein detection

• CRISPR/Cas applications:

  • CUT&RUN as an alternative to ChIP

  • CRISPR activation/inhibition for functional studies

  • Base editing for introducing specific mutations

These technologies, when combined with traditional antibody-based methods, can provide unprecedented insights into At1g61060 function and interactions.

What are the best practices for ensuring reproducibility when using At1g61060 Antibody?

To ensure reproducible research with At1g61060 Antibody:

• Comprehensive documentation:

  • Record antibody catalog number, lot number, and supplier

  • Document detailed experimental protocols including all buffer compositions

  • Maintain complete records of plant growth conditions and treatments

• Rigorous controls:

  • Include both positive and negative controls in all experiments

  • Use genetic controls (knockout/knockdown plants)

  • Implement technical and biological replicates (minimum n=3)

• Validation and verification:

  • Regularly validate antibody specificity, especially with new lots

  • Verify key findings using complementary approaches

  • Consider independent replication of critical experiments

• Transparent reporting:

  • Follow reporting guidelines for antibody-based research

  • Share detailed methods including all optimization steps

  • Provide access to raw data and analysis workflows

• Standardization efforts:

  • Use standardized protocols where available

  • Participate in community efforts to benchmark antibody performance

  • Consider contributing validation data to public repositories