At1g60970 Antibody

What is At1g60970 and why is it significant in plant research?

At1g60970 refers to a specific protein encoded by the At1g60970 gene in Arabidopsis thaliana (mouse-ear cress), an important model organism in plant biology. The protein (UniProt No. Q940S5) is studied to understand specific molecular pathways in plant development and stress responses. Research with At1g60970 antibody enables scientists to detect, quantify, and characterize this protein in experimental systems. Investigations typically focus on protein expression patterns across different plant tissues, developmental stages, or in response to various environmental stimuli. The antibody serves as a crucial tool for exploring protein localization, interaction networks, and functional roles within cellular contexts .

What are the key technical specifications of commercially available At1g60970 antibodies?

Commercial At1g60970 antibodies are typically polyclonal antibodies raised in rabbits using recombinant Arabidopsis thaliana At1g60970 protein as the immunogen. These antibodies are designed specifically for research applications with the following standard specifications:

These antibodies are typically validated for specificity using recombinant protein standards and wild-type Arabidopsis tissue samples .

How can I validate the specificity of At1g60970 antibody in my experiments?

Validating antibody specificity is crucial to ensure reliable research results. For At1g60970 antibody, implement the following comprehensive validation strategy:

• Genetic validation: Compare antibody reactivity in wild-type plants versus At1g60970 knockout mutants. True-positive signals should be absent or significantly reduced in knockout samples.

• Overexpression validation: Test the antibody on samples from plants overexpressing At1g60970. An increased signal intensity should correspond with elevated protein levels.

• Peptide competition assay: Pre-incubate the antibody with excess purified At1g60970 protein or immunizing peptide before application to samples. Specific signals should be blocked or diminished.

• Orthogonal validation: Correlate antibody-based detection with mRNA expression data from RT-PCR or RNA-seq experiments to confirm consistency.

• Western blot molecular weight verification: Confirm that the detected band appears at the expected molecular weight for At1g60970 protein.

Remember that antibody specificity issues are common in research. Several studies have demonstrated that commercial antibodies can exhibit non-specific binding patterns that lead to misidentification of target proteins . For instance, antibodies against the angiotensin II type 1 receptor showed identical staining patterns in both wild-type and receptor knockout mice, illustrating the critical importance of proper validation .

What controls should I implement when using At1g60970 antibody?

Implementing appropriate controls is essential for experimental rigor when working with At1g60970 antibody:

• Positive control: Include a sample known to express At1g60970 (e.g., specific Arabidopsis tissue where the protein is well-characterized).

• Negative control:

  • Primary antibody omission: Process samples without the At1g60970 antibody to identify non-specific binding from secondary antibodies.

  • Isotype control: Use an irrelevant antibody of the same isotype and concentration to identify non-specific binding.

  • Genetic negative control: Use tissue from At1g60970 knockout plants when available.

• Loading control: For Western blots, include detection of a housekeeping protein (e.g., actin, tubulin) to normalize expression levels.

• Recombinant protein standard: Include purified At1g60970 protein at known concentrations to establish a standard curve for quantification.

• Competitive inhibition control: Pre-incubate antibody with immunizing peptide to confirm signal specificity.

These controls should be implemented systematically across experimental replicates to ensure reproducibility and accuracy of your findings .

What are the optimal conditions for using At1g60970 antibody in Western blotting?

For optimal Western blot results with At1g60970 antibody, follow this methodological approach:

Sample preparation:

• Extract total protein from plant tissue using a buffer containing protease inhibitors to prevent degradation.

• Quantify protein concentration using Bradford or BCA assay.

• Denature proteins by boiling in Laemmli buffer (containing SDS and β-mercaptoethanol) for 5 minutes.

Gel electrophoresis and transfer:

• Load 20-50 μg protein per lane on a 10-12% SDS-PAGE gel.

• Include molecular weight markers to verify target size.

• Transfer proteins to a PVDF or nitrocellulose membrane (0.45 μm pore size) at 100V for 1 hour or 30V overnight at 4°C.

Immunoblotting protocol:

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

• Dilute At1g60970 antibody 1:500 to 1:1000 in blocking solution.

• Incubate membrane with primary antibody overnight at 4°C with gentle agitation.

• Wash 3 times for 10 minutes each with TBST.

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

• Wash 3 times for 10 minutes each with TBST.

• Develop using enhanced chemiluminescence (ECL) substrate and detect signal.

Expected result: At1g60970 protein should appear as a specific band at the predicted molecular weight. If multiple bands appear, additional optimization or validation may be necessary .

How can I optimize ELISA protocols using At1g60970 antibody?

ELISA optimization for At1g60970 detection requires methodical testing of various parameters:

Indirect ELISA protocol:

• Coating: Add 100 μL of antigen (plant extract or recombinant At1g60970) diluted in carbonate-bicarbonate buffer (pH 9.6) to each well. Incubate overnight at 4°C.

• Blocking: Add 300 μL of 3% BSA in PBS. Incubate for 1 hour at room temperature.

• Primary antibody: Add 100 μL of At1g60970 antibody diluted 1:500-1:2000 in 1% BSA/PBS. Incubate for 2 hours at room temperature.

• Secondary antibody: Add 100 μL of HRP-conjugated anti-rabbit antibody diluted 1:5000 in 1% BSA/PBS. Incubate for 1 hour at room temperature.

• Detection: Add 100 μL of TMB substrate. Incubate for 15-30 minutes protected from light.

• Stop reaction: Add 100 μL of 2N H₂SO₄ and read absorbance at 450 nm.

Optimization parameters:

• Test multiple antibody dilutions (1:500, 1:1000, 1:2000, 1:5000) to determine optimal concentration

• Compare different blocking agents (BSA, non-fat milk, commercial blockers)

• Evaluate incubation times (1-4 hours for primary antibody, 30-60 minutes for secondary)

• Test various washing protocols (3-5 washes for 5-10 minutes each)

For sandwich ELISA, consider both symmetrical and asymmetrical approaches as described in the literature. Asymmetrical assays using different capture and detection antibodies often provide higher specificity and sensitivity, especially when combining monoclonal capture antibodies with polyclonal detection antibodies .

Why might I observe multiple bands or non-specific signals when using At1g60970 antibody?

Multiple bands or non-specific signals are common challenges when working with antibodies. For At1g60970 antibody, several factors may contribute to this issue:

Potential causes and solutions:

• Post-translational modifications: At1g60970 may exist in different forms due to phosphorylation, glycosylation, or other modifications.

  • Solution: Use phosphatase or glycosidase treatments to confirm if additional bands represent modified forms.

• Protein degradation: Partial degradation of the target protein can result in fragments detected by the antibody.

  • Solution: Use fresher samples, add additional protease inhibitors, and keep samples cold throughout preparation.

• Cross-reactivity: The antibody may recognize epitopes present in other proteins.

  • Solution: Perform peptide competition assays to identify specific versus non-specific bands.

• Non-specific antibody binding: The literature shows that even commercial antibodies can exhibit significant non-specific binding patterns .

  • Solution: Increase blocking concentration (5-10% BSA or milk), optimize antibody dilution, and increase washing stringency.

• Alternative splicing: Different isoforms of At1g60970 may be expressed.

  • Solution: Compare band patterns with known splice variant sizes and validate with RT-PCR.

A study investigating antibodies against the angiotensin type 1 receptor found that commercial antibodies showed identical band patterns in both wild-type and knockout mice, demonstrating significant specificity issues . This highlights the importance of rigorous validation, especially when unexpected band patterns are observed.

How can I differentiate between genuine results and artifacts when interpreting At1g60970 antibody data?

Distinguishing true signals from artifacts requires a systematic approach to data interpretation:

• Consistent molecular weight: The primary band for At1g60970 should appear at the predicted molecular weight. Significant deviation suggests potential non-specific binding.

• Consistency across methods: Compare results from different detection techniques (Western blot, ELISA, immunohistochemistry). True results should be consistent across multiple methodologies.

• Biological reproducibility: Results should be reproducible across biological replicates. Inconsistent patterns may indicate technical artifacts.

• Correlation with orthogonal data: At1g60970 protein levels should generally correlate with mRNA expression data from RT-PCR or RNA-seq experiments.

• Genetic validation: Compare signal intensity between wild-type and knockout/knockdown samples. True signals should be reduced or eliminated in samples with reduced target expression.

• Technical validation: Include all appropriate controls as described in section 2.2.

Research has demonstrated that apparent positive antibody staining can appear in tissues completely lacking the target protein . For instance, immunohistochemical staining in the liver vasculature of receptor knockout mice was observed despite the confirmed absence of the target receptor, highlighting how convincing artifacts can be without proper validation .

How can I use At1g60970 antibody for studying protein-protein interactions?

At1g60970 antibody can be instrumental in investigating protein interaction networks through several advanced techniques:

Co-immunoprecipitation (Co-IP) protocol:

• Prepare plant lysate in non-denaturing lysis buffer (typically 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors).

• Pre-clear lysate with Protein A/G beads for 1 hour at 4°C.

• Incubate pre-cleared lysate with At1g60970 antibody (2-5 μg) overnight at 4°C with gentle rotation.

• Add Protein A/G beads and incubate for 2-4 hours at 4°C.

• Wash beads 4-5 times with wash buffer (lysis buffer with reduced detergent).

• Elute bound proteins by boiling in Laemmli buffer.

• Analyze interacting proteins by SDS-PAGE followed by:

  • Silver staining for total protein visualization

  • Western blotting for specific suspected interactors

  • Mass spectrometry for unbiased identification of all co-precipitated proteins

Proximity-dependent biotin identification (BioID):
By fusing a biotin ligase to At1g60970, you can identify proximal proteins that get biotinylated in vivo, which can then be isolated using streptavidin beads and identified using mass spectrometry. The At1g60970 antibody can be used to confirm the expression and proper localization of the fusion protein.

When interpreting protein-protein interaction data, consider that:

• Some interactions may be indirect through larger complexes

• Transient interactions might be difficult to capture

• Buffer conditions can significantly influence interaction stability

• Overexpression systems may create artificial interactions

Validation of interactions through orthogonal methods such as yeast two-hybrid, FRET, or BiFC is recommended to confirm true biological interactions .

What approaches can I use to study post-translational modifications of At1g60970?

Investigating post-translational modifications (PTMs) of At1g60970 requires specialized techniques:

Identification of phosphorylation sites:

• Immunoprecipitate At1g60970 using the validated antibody.

• Separate proteins by SDS-PAGE and excise the At1g60970 band.

• Perform in-gel digestion with trypsin.

• Analyze peptides by LC-MS/MS with a focus on phosphopeptide enrichment.

• Confirm phosphorylation sites using phosphatase treatment:

  • Split your sample and treat half with lambda phosphatase

  • Compare mobility shifts in Western blots using At1g60970 antibody

Detection of ubiquitination:

• Perform immunoprecipitation with At1g60970 antibody.

• Probe Western blots with anti-ubiquitin antibodies to detect ubiquitinated forms.

• For in vivo studies, treat plants with proteasome inhibitors (MG132) to accumulate ubiquitinated proteins before analysis.

Analysis of other modifications:

• For glycosylation: Treat samples with specific glycosidases before Western blotting with At1g60970 antibody to observe mobility shifts.

• For SUMOylation: Perform immunoprecipitation followed by Western blotting with anti-SUMO antibodies.

Generation of modification-specific antibodies:
If a specific PTM site is identified and of particular interest, consider developing modification-specific antibodies using synthetic peptides containing the modified residue. These can be particularly valuable for studying the regulation and function of At1g60970 in different physiological contexts.

When working with PTMs, it's essential to use appropriate controls and inhibitor cocktails during sample preparation to preserve the modifications of interest, as they can be labile during processing .

How can I generate and utilize At1g60970 antibody fragments for specialized research applications?

Antibody fragmentation creates smaller antibody derivatives with unique properties that can be advantageous for certain applications. For At1g60970 antibody, pepsin digestion can be used to generate F(ab')₂ fragments:

Pepsin digestion protocol:

• Prepare the following materials:

  • 25-35 mg of At1g60970 antibody

  • 10 mg Pepsin from porcine gastric mucosa

  • 0.1M sodium citrate buffer pH 3.5

  • 5N NaOH

  • 2M TRIS-base

  • PBS (0.01M, pH 7.2-7.4)

• Dialyze antibody against 0.1M sodium citrate buffer (pH 3.5).

• Add pepsin at a ratio of 1:20 to 1:50 (pepsin:antibody) by weight.

• Incubate at 37°C for 4-24 hours (optimum time should be determined empirically).

• Monitor digestion progress using SDS-PAGE to track fragment generation.

• Stop digestion by adjusting pH to 7.0 with 2M Tris or 5N NaOH.

• Purify F(ab')₂ fragments using protein A affinity chromatography (fragments will flow through while undigested antibodies and Fc fragments will bind).

Applications of antibody fragments:

• Reduced non-specific binding: The absence of Fc regions reduces binding to Fc receptors present on many cell types.

• Enhanced tissue penetration: Smaller fragments can penetrate tissues more efficiently for immunohistochemistry applications.

• Reduced background in immunoassays: Particularly useful when detecting plant proteins that may have endogenous Fc-binding proteins.

• Specialized immunoprecipitation: When working with samples containing proteins that bind to the Fc region of antibodies.

SDS-PAGE and Western blot analysis can confirm successful fragmentation while preserving antigen binding activity, as demonstrated in time-course analyses of antibody digestion .

What advanced imaging techniques can I use with At1g60970 antibody for subcellular localization studies?

Advanced imaging with At1g60970 antibody can provide valuable insights into protein localization and dynamics:

Immunofluorescence microscopy protocol:

• Fix plant tissue samples in 4% paraformaldehyde in PBS for 30 minutes.

• Permeabilize with 0.1% Triton X-100 in PBS for 15 minutes.

• Block with 3% BSA in PBS for 1 hour.

• Incubate with At1g60970 antibody (1:100-1:500 dilution) overnight at 4°C.

• Wash 3 times with PBS for 5 minutes each.

• Incubate with fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488 anti-rabbit) for 1 hour at room temperature.

• Counterstain with DAPI (1 μg/mL) for nuclear visualization.

• Mount slides with anti-fade mounting medium.

Super-resolution microscopy techniques:

• Structured Illumination Microscopy (SIM): Achieves resolution of ~100 nm through computational reconstruction of patterned illumination images.

• Stimulated Emission Depletion (STED): Uses a depletion laser to reduce the effective fluorescence area, achieving resolution of ~30-80 nm.

• Photoactivated Localization Microscopy (PALM)/Stochastic Optical Reconstruction Microscopy (STORM): Single-molecule localization techniques achieving resolutions of ~10-20 nm.

Proximity Ligation Assay (PLA):
This technique can detect protein-protein interactions with spatial resolution:

• Incubate fixed samples with At1g60970 antibody and an antibody against a suspected interacting protein.

• Add PLA probes (secondary antibodies with attached oligonucleotides).

• If proteins are in close proximity (<40 nm), the oligonucleotides can be ligated and amplified.

• Detect amplified signal as distinct fluorescent spots.

For all imaging applications, include appropriate controls as discussed in section 2.2, and consider z-stack acquisition for three-dimensional analysis of protein distribution within cellular structures .

How can I integrate At1g60970 antibody research with systems biology approaches?

Integrating At1g60970 antibody research with systems biology creates opportunities for comprehensive understanding of protein function in complex biological contexts:

Multi-omics integration strategies:

• Proteomics correlation: Compare At1g60970 antibody-based quantification across different conditions with global proteomics data to identify co-regulated proteins.

• Transcriptomics integration: Correlate At1g60970 protein levels with gene expression data to identify post-transcriptional regulation mechanisms.

• Metabolomics connection: Analyze changes in metabolic profiles in relation to At1g60970 protein levels to identify functional metabolic connections.

Network analysis approaches:

• Use At1g60970 antibody for co-immunoprecipitation followed by mass spectrometry to identify protein interaction networks.

• Map identified interactions to known signaling and metabolic pathways.

• Perform enrichment analysis to identify overrepresented biological processes.

• Visualize networks using tools like Cytoscape with At1g60970 as a central node.

Spatial proteomics applications:

• Combine At1g60970 antibody-based detection with cell fractionation to analyze protein distribution across subcellular compartments.

• Integrate spatial information with protein interaction data to build compartment-specific interaction networks.

• Study dynamic changes in localization under different environmental conditions or developmental stages.

This integrated approach enables the placement of At1g60970 within the broader context of cellular physiology, revealing emergent properties that might not be apparent through isolated antibody-based studies alone. The systems-level perspective can guide hypothesis generation for further targeted experiments using At1g60970 antibody as a specific probe .

What are the latest methodological advances in antibody-based detection relevant to At1g60970 research?

Recent methodological advances have expanded the capabilities of antibody-based detection systems with potential applications to At1g60970 research:

Single-cell proteomics approaches:

• Imaging Mass Cytometry: Combines antibody specificity with mass spectrometry detection for highly multiplexed single-cell analysis.

• CyTOF (Mass Cytometry): Uses metal-tagged antibodies for simultaneous detection of multiple proteins at single-cell resolution.

• Single-cell Western blotting: Allows protein quantification in individual cells using specialized microfluidic platforms.

Advanced microfluidics applications:

• Digital ELISA: Enables detection of proteins at femtomolar concentrations through single-molecule counting.

• Droplet-based immunoassays: Provides high-throughput analysis with minimal sample consumption.

Computational advancements:

• Deep learning for image analysis: Enhances detection and quantification from immunofluorescence images.

• Antibody binding prediction algorithms: Helps design optimal epitope targeting for new antibody development.

Innovative detection methods:

• Electrochemical immunosensors: Offer label-free detection with high sensitivity.

• Surface plasmon resonance imaging: Provides real-time, label-free detection of biomolecular interactions.

• Lanthanide-based time-resolved fluorescence: Reduces background interference through time-gated detection.

Antibody engineering:

• Nanobodies (VHH antibodies): Single-domain antibody fragments offering improved penetration and stability.

• Bispecific antibodies: Recognize two different epitopes simultaneously, enabling novel detection strategies.

These technological advances can be applied to At1g60970 research to achieve higher sensitivity, specificity, and throughput in protein detection, potentially revealing previously undetectable aspects of At1g60970 biology in plant systems .