At2g21120 Antibody

What is At2g21120 and why are antibodies against it important for research?

At2g21120 is a gene identifier for a protein found in Arabidopsis thaliana, located on chromosome 2. Antibodies targeting this protein are valuable tools for studying plant molecular biology, particularly in research involving protein detection, localization, and functional studies. These antibodies enable researchers to investigate protein expression patterns, protein-protein interactions, and biochemical pathways involving At2g21120 . Recent advances in de novo antibody design have enabled the development of precise antibodies against targets like At2g21120 without requiring prior antibody information, significantly expanding research capabilities in this area.

What types of antibodies are available for At2g21120 detection?

Researchers typically have access to both polyclonal and monoclonal antibodies for At2g21120 detection. Polyclonal antibodies recognize multiple epitopes on the target protein, offering higher sensitivity but potentially lower specificity. Monoclonal antibodies, in contrast, recognize a single epitope, providing higher specificity but potentially lower sensitivity. Recent advances in computational antibody design, as demonstrated in the 2025 study on de novo antibody design, have enabled the creation of highly specific monoclonal antibodies for various protein targets including At2g21120 . These computationally designed antibodies are showing promise for distinguishing closely related protein subtypes with high molecular specificity.

How should I validate an At2g21120 antibody before experimental use?

Proper validation of At2g21120 antibodies should follow a multi-step process:

• Western blot analysis: Confirm the antibody detects a band of the expected molecular weight (specific to At2g21120 protein)

• Immunoprecipitation: Verify the antibody can pull down the target protein from cell lysates

• Immunofluorescence/Immunohistochemistry: Ensure proper localization patterns consistent with known distribution

• Negative controls: Test antibody in systems where At2g21120 is knocked down or absent

• Cross-reactivity analysis: Assess potential cross-reactivity with similar proteins

Methods such as dot-blot analysis can also be valuable for assessing antibody specificity, similar to techniques used in other antibody validation studies . Proper validation ensures experimental reliability and reproducibility in subsequent research applications.

What are the most effective methods for generating At2g21120-specific antibodies?

Recent advances have revolutionized antibody production for targets like At2g21120. Traditional methods include:

• Hybridoma technology: Generation of monoclonal antibodies through fusion of antibody-producing B cells with myeloma cells

• Phage display: Selection of antibody fragments from large libraries displayed on bacteriophage

• Recombinant antibody production: Expression of engineered antibodies in various host systems

• Creating approximately 10^6 sequence combinations

• Combining 10^2 designed light chain sequences with 10^4 designed heavy chain sequences

• Using yeast display technology to identify high-affinity binders

• Converting promising candidates to full IgG format for further characterization

This computational design approach has demonstrated unprecedented precision compared to previous de novo antibody design reports, enabling discovery of antibodies with varying binding strengths for all six tested targets, including cases without experimentally resolved target structures .

How can I improve the specificity of At2g21120 antibodies?

Enhancing the specificity of At2g21120 antibodies can be achieved through several methodological approaches:

• Epitope selection: Choose unique epitopes with minimal homology to related proteins

• Affinity maturation: Introduce targeted mutations to increase binding affinity and specificity

• Negative selection: Screen antibody candidates against related proteins to eliminate cross-reactive clones

• Computational design: Utilize structure-based predictions to identify optimal binding interfaces

The recent advances in computational antibody design have demonstrated remarkable success in generating antibodies capable of distinguishing closely related protein subtypes or mutants . This approach leverages atomic-accuracy structure prediction to design antibodies with tailored properties, achieving high molecular specificity that can differentiate subtle variations in target proteins.

What are the advantages of monoclonal versus polyclonal antibodies for At2g21120 research?

The choice between monoclonal and polyclonal antibodies for At2g21120 research depends on specific experimental needs:

For At2g21120 research requiring precise distinction between closely related plant proteins, computationally designed monoclonal antibodies offer superior specificity similar to the antibodies described in the 2025 study, which demonstrated the ability to distinguish closely related protein subtypes .

What are the optimal conditions for using At2g21120 antibodies in western blotting?

Optimal western blotting conditions for At2g21120 antibodies typically include:

• Sample preparation:

  • Use fresh plant tissue or cell culture material

  • Extract proteins in buffer containing protease inhibitors

  • Heat samples at 95°C for 5 minutes in reducing SDS sample buffer

• Gel electrophoresis and transfer:

  • 10-12% SDS-PAGE for optimal resolution

  • Semi-dry or wet transfer to nitrocellulose membrane (0.45 μm pore size)

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in PBS for 1 hour at room temperature

  • Primary antibody dilution: 1:1000 to 1:5000 in 1% PBS-milk solution

  • Incubate primary antibody for 1-2 hours at room temperature or overnight at 4°C

  • Secondary antibody (HRP-conjugated): 1:5000 to 1:10000 dilution

  • Detection using enhanced chemiluminescence substrate

These protocols are adapted from established antibody validation methods, similar to those described for other research antibodies . Optimization may be necessary based on the specific properties of the At2g21120 antibody being used.

How can I optimize immunoprecipitation protocols for At2g21120?

For successful immunoprecipitation of At2g21120, consider the following methodological approach:

• Lysate preparation:

  • Harvest plant tissue or cells and lyse in non-denaturing buffer

  • Use buffer containing 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1% NP-40 or Triton X-100

  • Include protease and phosphatase inhibitors freshly before use

  • Clear lysate by centrifugation (10,000 g for 10 minutes at 4°C)

• Antibody binding:

  • Pre-clear lysate with protein A/G beads (30 min at 4°C)

  • Add 2-5 μg of At2g21120 antibody per 500 μg of protein lysate

  • Incubate with rotation overnight at 4°C

  • Add pre-washed protein A/G beads and incubate 2-4 hours at 4°C

• Washing and elution:

  • Wash beads 4-5 times with cold IP buffer

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

• Analysis:

  • Perform SDS-PAGE and western blot to confirm successful IP

  • Consider mass spectrometry for interactome analysis

This protocol is designed based on successful IP methodologies used for other research antibodies, as demonstrated in previous studies evaluating antibody suitability for immunoprecipitation assays .

What controls should I include when performing immunofluorescence with At2g21120 antibodies?

Proper immunofluorescence experiments with At2g21120 antibodies require rigorous controls:

• Essential negative controls:

  • Secondary antibody only (omit primary antibody)

  • Isotype control (irrelevant primary antibody of same isotype)

  • Pre-immune serum (for polyclonal antibodies)

  • Tissue/cells lacking At2g21120 expression (genetic knockout if available)

• Blocking peptide control:

  • Pre-incubate antibody with excess immunizing peptide

  • Should abolish specific staining

• Positive controls:

  • Tissue/cells known to express At2g21120

  • Co-staining with another marker of the expected subcellular compartment

• Technical validation:

  • Multiple fixation methods comparison

  • Different antibody concentrations testing

  • Comparison of results with alternative At2g21120 antibodies if available

Including these controls ensures that observed signals are specific to At2g21120 rather than artifacts or non-specific binding, similar to validation approaches used for other antibodies in research settings .

How can I use At2g21120 antibodies for chromatin immunoprecipitation (ChIP) experiments?

For effective ChIP experiments using At2g21120 antibodies, follow this methodological approach:

• Sample preparation:

  • Crosslink plant tissue with 1% formaldehyde for 10 minutes

  • Quench with 0.125 M glycine for 5 minutes

  • Isolate nuclei and perform sonication to generate chromatin fragments (200-500 bp)

  • Reserve 5-10% of sheared chromatin as input control

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Add 3-5 μg of At2g21120 antibody to 25-50 μg of chromatin

  • Incubate overnight at 4°C with rotation

  • Add protein A/G beads and incubate 2-4 hours at 4°C

  • Perform stringent washing steps

• Crosslink reversal and DNA purification:

  • Reverse crosslinks by heating at 65°C overnight

  • Treat with RNase A and Proteinase K

  • Purify DNA using column-based methods

• Analysis:

  • Perform qPCR targeting regions of interest

  • Consider ChIP-seq for genome-wide binding analysis

The success of ChIP experiments depends heavily on antibody quality. Antibodies developed through computational design approaches similar to those described in the 2025 study may offer superior specificity for distinguishing closely related protein isoforms in complex chromatin environments .

How can RNA-binding protein immunoprecipitation (RIP) be performed with At2g21120 antibodies?

RIP assays are valuable for studying RNA-protein interactions involving At2g21120. The methodology includes:

• Cell/tissue preparation:

  • Harvest plant tissue or cells in non-denaturing conditions

  • Homogenize in lysis buffer containing RNase inhibitors

  • Clear lysate by centrifugation

• Immunoprecipitation:

  • Pre-clear lysate with protein A/G beads

  • Add 5 μg of At2g21120 antibody

  • Incubate with rotation overnight at 4°C

  • Add protein A/G beads and incubate for 3 hours at 4°C

  • Wash beads thoroughly with RIP wash buffer

• RNA isolation and analysis:

  • Extract RNA from immunoprecipitated material

  • Treat with DNase to remove contaminating DNA

  • Perform RT-qPCR or RNA-seq to identify bound RNAs

• Controls and validation:

  • IgG control immunoprecipitation

  • Input RNA control

  • Validation of enriched transcripts by independent methods

This approach is based on established RIP protocols that have been successfully applied to other RNA-binding proteins, similar to those described for TLS/FUS protein studies . For At2g21120, the specificity of computationally designed antibodies would be particularly advantageous for distinguishing between closely related RNA-binding proteins that might have overlapping targets.

What strategies can overcome epitope masking in At2g21120 detection?

Epitope masking can prevent antibody recognition of At2g21120 due to protein conformation, post-translational modifications, or protein-protein interactions. To overcome these challenges:

• Sample preparation modifications:

  • Test multiple extraction buffers with different detergents

  • Try various fixation methods for immunostaining

  • Consider antigen retrieval techniques:

    • Heat-induced epitope retrieval (100°C in citrate buffer, pH 6.0)

    • Enzymatic retrieval (proteinase K treatment)

    • Strong denaturing conditions for western blotting

• Alternative antibody approaches:

  • Use antibodies targeting different epitopes on At2g21120

  • Consider using both polyclonal and monoclonal antibodies

  • Develop computationally designed antibodies targeting less-masked regions

• Experimental adaptations:

  • For protein complexes: use crosslinking to stabilize interactions

  • For post-translational modifications: use phosphatase or deglycosylase treatments

  • For membrane proteins: optimize membrane protein extraction protocols

The computational antibody design approach described in the 2025 study could be particularly valuable for developing antibodies targeting accessible epitopes of At2g21120 , as the atomic-level structural predictions can identify optimal binding sites even in challenging protein conformations.

Why might western blots with At2g21120 antibodies show multiple bands, and how should this be interpreted?

Multiple bands in western blots using At2g21120 antibodies could result from several biological or technical factors:

To distinguish between these possibilities:

• Compare observed band patterns with predicted molecular weights of known isoforms

• Perform peptide competition assays to determine which bands are specific

• Use genetic knockdown/knockout samples as negative controls

• Consider alternative antibodies targeting different epitopes of At2g21120

This analytical approach follows established antibody validation methodologies used in other protein studies .

How can I improve signal-to-noise ratio when using At2g21120 antibodies?

Optimizing signal-to-noise ratio requires systematic approach to both increase specific signal and reduce background:

• Enhancing specific signal:

  • Optimize antibody concentration through titration experiments

  • Adjust incubation time and temperature

  • Use signal amplification systems (tyramide signal amplification for IHC/IF)

  • Consider more sensitive detection systems (enhanced chemiluminescence substrates)

• Reducing background noise:

  • Increase blocking stringency (5% BSA or 5% milk, add 0.1-0.3% Tween-20)

  • Extend blocking time (overnight at 4°C)

  • Add carrier proteins to antibody diluent (0.5-1% BSA)

  • Use more stringent washing (increase number, duration, and detergent concentration)

  • Pre-absorb antibodies with tissues lacking At2g21120

• Antibody quality considerations:

  • Use affinity-purified antibodies when possible

  • Consider computationally designed antibodies with high specificity

  • Store antibodies properly to prevent degradation

Systematic optimization of these parameters, particularly when working with novel targets like At2g21120, is essential for achieving reliable results in different experimental contexts.

What are the most effective strategies for quantitative analysis of At2g21120 using antibody-based methods?

For accurate quantitative analysis of At2g21120, consider these methodological approaches:

• Western blot quantification:

  • Use internal loading controls (housekeeping proteins)

  • Include standard curves with recombinant At2g21120 protein

  • Apply appropriate normalization methods

  • Use modern digital imaging systems rather than film

  • Ensure signal is within linear detection range

• ELISA development:

  • Develop sandwich ELISA using two antibodies recognizing different epitopes

  • Include standard curves with purified At2g21120 protein

  • Validate assay for specificity, sensitivity, and reproducibility

  • Determine lower limit of detection and quantification

• Flow cytometry:

  • Establish proper controls for gating and fluorescence compensation

  • Use median fluorescence intensity for quantification

  • Include calibration beads to standardize measurements

  • Consider dual staining to normalize for cell size/protein content

• Automated image analysis for immunofluorescence/IHC:

  • Use standardized acquisition settings

  • Apply thresholding consistently across samples

  • Quantify signal intensity, area, or colocalization coefficients

  • Include reference standards in each experiment

These quantitative approaches should be validated with appropriate positive and negative controls, similar to the methodological validation described for other antibody-based techniques .

How can computational antibody design improve At2g21120 antibody development?

Computational antibody design represents a revolutionary approach for developing highly specific At2g21120 antibodies:

• Current capabilities:

  • The 2025 study demonstrated de novo antibody design without prior antibody information

  • Successfully created binders for six distinct target proteins

  • Generated 10^6 candidate sequences by combining designed light and heavy chains

  • Identified binders with varying binding strengths for all targets

  • Achieved specificity capable of distinguishing closely related protein subtypes

• Advantages for At2g21120 research:

  • Can design antibodies even without experimental structures of At2g21120

  • Enables targeting of specific epitopes for optimal detection

  • Allows rational design of antibodies that distinguish between closely related plant proteins

  • Produces antibodies with tailored properties (affinity, specificity, developability)

• Future potential:

  • Integration with AI-powered prediction of protein interactions

  • Design of antibodies targeting specific post-translational modifications

  • Engineering antibodies with enhanced tissue penetration for in vivo applications

  • Development of bispecific antibodies for studying protein complexes

This computational approach holds tremendous promise for creating next-generation research tools for studying challenging targets like At2g21120 .

What are the applications of At2g21120 antibodies in multiplex imaging techniques?

Advanced multiplex imaging with At2g21120 antibodies enables sophisticated spatial biology studies:

• Cyclic immunofluorescence (CycIF):

  • Sequential staining, imaging, and signal removal

  • Can incorporate At2g21120 antibodies with dozens of other markers

  • Enables colocalization studies with subcellular resolution

  • Requires careful antibody validation for multiplexing compatibility

• Mass cytometry imaging (IMC):

  • Labels At2g21120 antibodies with rare earth metals

  • Allows simultaneous detection of 40+ proteins

  • Provides single-cell resolution in tissue context

  • Requires specialized equipment and metal-conjugated antibodies

• DNA-barcoded antibody methods:

  • Conjugates At2g21120 antibodies with unique DNA oligonucleotides

  • Enables highly multiplexed detection through sequential hybridization

  • Can be combined with RNA detection for multi-omic analyses

  • Requires validation of barcode conjugation effects on antibody binding

• Practical considerations:

  • Validate antibody performance in multiplexed formats

  • Confirm absence of cross-reactivity with other primary antibodies

  • Optimize signal amplification for low-abundance targets

  • Implement computational analysis pipelines for multi-dimensional data

These approaches enable unprecedented insights into At2g21120 protein localization and interaction networks within complex cellular contexts, particularly when using computationally designed antibodies with high specificity .

How can RNA-protein interaction studies benefit from At2g21120 antibodies?

At2g21120 antibodies enable sophisticated analysis of RNA-protein interactions through several advanced methodologies:

• RNA-binding protein immunoprecipitation (RIP):

  • Immunoprecipitates At2g21120 with associated RNAs

  • Allows identification of direct RNA targets

  • Can be combined with high-throughput sequencing (RIP-seq)

  • Requires careful optimization to preserve RNA-protein interactions

• Cross-linking immunoprecipitation (CLIP):

  • Utilizes UV cross-linking to stabilize RNA-protein interactions

  • Provides higher stringency than standard RIP

  • Enables mapping of binding sites with nucleotide resolution

  • Requires highly specific antibodies like those developed through computational design

• Proximity ligation assays:

  • Combines At2g21120 antibodies with RNA probes

  • Detects RNA-protein interactions in situ

  • Provides spatial context for interactions

  • Requires optimization of fixation and permeabilization conditions

• RNA-protein interaction screening:

  • Uses At2g21120 antibodies to identify RNA binding partners

  • Can incorporate RNA libraries or cellular transcriptomes

  • Enables discovery of novel regulatory interactions

  • Benefits from highly specific antibodies to reduce false positives

These methodologies provide complementary insights into At2g21120's RNA binding properties and regulatory functions, with each approach offering distinct advantages for specific research questions. The importance of antibody specificity for RNA-binding protein studies has been demonstrated in previous research , highlighting the potential value of computationally designed antibodies for these applications.