At4g23960 Antibody

What is At4g23960 and what role does it play in Arabidopsis thaliana?

At4g23960 is a gene that encodes a probable F-box protein in Arabidopsis thaliana (Mouse-ear cress). It is also known by the alternative identifiers T32A16.130 and T32A16_130 . F-box proteins generally function as part of the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex, which is involved in protein ubiquitination and subsequent degradation via the 26S proteasome pathway. This process is crucial for regulating various cellular processes including cell cycle progression, signal transduction, and developmental pathways in plants. The specific functional role of the At4g23960 F-box protein in Arabidopsis remains an active area of research, with potential implications for plant growth regulation and stress responses.

What are the available formats of At4g23960 antibodies for research applications?

Based on available research resources, At4g23960 antibodies are primarily available as rabbit-derived polyclonal antibodies raised against Arabidopsis thaliana . These antibodies are specifically designed to recognize and bind to the probable F-box protein encoded by the At4g23960 gene. Additionally, there are recombinant protein options available for generating custom antibodies or for use as positive controls in immunoassays. The recombinant protein can be produced in various expression systems including E. coli, yeast, baculovirus, or mammalian cell systems, with a purity typically greater than or equal to 85% as determined by SDS-PAGE .

What are the verified applications for At4g23960 antibodies in plant research?

At4g23960 antibodies have been validated for several experimental applications in plant research:

• Western Blot (WB): Useful for detecting the target protein in plant tissue lysates and confirming protein expression levels or post-translational modifications.

• Enzyme-Linked Immunosorbent Assay (ELISA): Enables quantitative measurement of At4g23960 protein levels in plant samples .

While not explicitly mentioned in the search results, antibodies of this nature may also potentially be used for:

• Immunoprecipitation (IP): To isolate protein complexes containing the At4g23960 protein.

• Immunohistochemistry (IHC): To visualize protein localization in plant tissue sections.

• Chromatin Immunoprecipitation (ChIP): If the protein functions in transcriptional regulation.

How should researchers validate the specificity of At4g23960 antibodies for experimental use?

Validating antibody specificity is crucial for obtaining reliable research results. For At4g23960 antibodies, researchers should implement a multi-step validation process:

• Positive and negative controls: Include wild-type Arabidopsis samples (positive control) and knockout/knockdown lines of At4g23960 (negative control) when performing immunoblotting.

• Pre-adsorption tests: Pre-incubate the antibody with purified recombinant At4g23960 protein before immunodetection. Specific antibodies will show reduced or absent signal when pre-adsorbed.

• Cross-reactivity assessment: Test the antibody against closely related F-box proteins to ensure it doesn't cross-react with other family members.

• Multiple antibodies approach: When possible, use multiple antibodies targeting different epitopes of the At4g23960 protein to confirm findings.

• Mass spectrometry validation: Confirm identity of immunoprecipitated proteins using mass spectrometry to verify the antibody is capturing the intended target.

Similar methodological approaches have been used for validating antibodies in other biological systems, as demonstrated in the development of the A4 antibody for neuraminidase detection, where dot-blot tests were employed to confirm binding specificity .

What are optimal conditions for using At4g23960 antibodies in Western blot experiments?

Based on general practices for plant F-box protein detection and the known applications of At4g23960 antibodies, the following protocol is recommended:

Sample Preparation:

• Extract proteins from plant tissues using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitor cocktail

• Sonicate briefly and centrifuge at 14,000g for 15 minutes at 4°C

• Quantify protein concentration using Bradford or BCA assay

SDS-PAGE and Transfer:

• Load 20-50μg of total protein per lane

• Separate proteins on 10-12% SDS-PAGE gels

• Transfer to PVDF membrane at 100V for 60 minutes in cold transfer buffer

Immunoblotting:

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

• Incubate with At4g23960 antibody at 1:1000 dilution in TBST with 1% BSA overnight at 4°C

• Wash 3x with TBST, 10 minutes each

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

• Wash 3x with TBST, 10 minutes each

• Develop using ECL substrate and image

Expected Results:

• The predicted molecular weight of At4g23960 is approximately 45-50 kDa, but this may vary depending on post-translational modifications

How can researchers use At4g23960 antibodies to study protein-protein interactions in the SCF complex?

F-box proteins like At4g23960 function within SCF complexes, making protein-protein interaction studies particularly relevant. Several methodological approaches can be employed:

• Co-immunoprecipitation (Co-IP):

  • Lyse plant tissues in non-denaturing buffer

  • Pre-clear lysate with Protein A/G beads

  • Incubate cleared lysate with At4g23960 antibody overnight

  • Capture antibody-protein complexes with Protein A/G beads

  • Wash and elute proteins for analysis by Western blot or mass spectrometry

  • Probe for known SCF components (ASK1/Skp1, Cullin1, Rbx1)

• Proximity Ligation Assay (PLA):

  • Fix and permeabilize plant cells/tissues

  • Incubate with At4g23960 antibody and antibody against potential interacting partner

  • Apply secondary antibodies with attached DNA probes

  • If proteins interact, DNA probes will be in close proximity

  • Ligation and amplification steps will generate a fluorescent signal viewable by microscopy

• Bimolecular Fluorescence Complementation (BiFC):

  • This requires cloning rather than antibodies directly but can validate interactions identified with antibody-based methods

  • Clone At4g23960 and potential interacting partners with split fluorescent protein fragments

  • Co-express in plant cells and observe for reconstituted fluorescence

What controls should be included when conducting immunodetection experiments with At4g23960 antibodies?

Proper experimental controls are critical for antibody-based research. For At4g23960 antibody experiments, researchers should include:

Positive Controls:

• Recombinant At4g23960 protein at known concentrations

• Wild-type Arabidopsis samples known to express At4g23960

• Samples with overexpressed At4g23960 (when available)

Negative Controls:

• At4g23960 knockout or knockdown plant lines

• Non-transformed wild-type samples for comparison with transgenic lines

• Primary antibody omission control to detect non-specific binding of secondary antibody

• Isotype control using non-specific rabbit IgG at the same concentration as the primary antibody

Specificity Controls:

• Pre-adsorption of antibody with recombinant antigen

• Western blot with recombinant At4g23960 protein to confirm recognition

• Dot blot analysis using various concentrations of purified protein

Loading Controls:

• Antibodies against housekeeping proteins (e.g., actin, tubulin, or GAPDH)

• Total protein staining methods (e.g., Ponceau S, SYPRO Ruby)

Similar control strategies have proven effective in other antibody development studies, such as the A4 antibody development for detection of neuraminidase variants .

How can At4g23960 antibodies be used to study protein expression under different environmental conditions?

At4g23960 antibodies can be valuable tools for studying how F-box protein expression changes in response to environmental stimuli. A recommended experimental approach includes:

Experimental Design:

• Subject Arabidopsis plants to different environmental conditions (e.g., drought, salt stress, temperature extremes, pathogen exposure)

• Collect tissue samples at multiple time points (0h, 6h, 12h, 24h, 48h)

• Extract proteins using consistent methodology

• Analyze protein expression by Western blot or ELISA using At4g23960 antibodies

Quantification Methods:

• Western blot:

  • Use digital image analysis software to quantify band intensity

  • Normalize to loading controls

  • Present as fold-change relative to time zero or control conditions

• ELISA:

  • Develop standard curves using recombinant At4g23960 protein

  • Calculate absolute protein concentrations in samples

  • Normalize to total protein content

Data Presentation:
Present results in a table format similar to the example below:

What are the key considerations for using At4g23960 antibodies in immunolocalization studies?

Immunolocalization can provide valuable insights into the subcellular localization and functional context of At4g23960. Key methodological considerations include:

Sample Preparation:

• Fixation: Use 4% paraformaldehyde in PBS for 1-2 hours for tissue preservation

• Embedding: Embed fixed tissues in paraffin or freeze in OCT compound

• Sectioning: Prepare 5-10μm sections on adhesive slides

Immunolabeling Protocol:

• Antigen Retrieval: Heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Blocking: 5% normal goat serum in PBS with 0.1% Triton X-100 for 1 hour

• Primary Antibody: Incubate with At4g23960 antibody (1:100-1:500 dilution) overnight at 4°C

• Secondary Antibody: Fluorescent-conjugated anti-rabbit IgG (1:500) for 1 hour

• Counterstaining: DAPI for nuclei visualization

• Mounting: Anti-fade mounting medium

Controls and Validation:

• Include sections from At4g23960 knockout plants as negative controls

• Perform co-localization studies with markers for relevant subcellular compartments

• Consider dual-labeling with antibodies against other SCF complex components

Advanced Applications:

• Super-resolution microscopy: For detailed subcellular localization

• Live-cell imaging: Using GFP-tagged proteins to complement antibody-based fixed-cell studies

• FRET analysis: To study protein-protein interactions in situ

How should researchers quantitatively analyze At4g23960 protein levels across different experimental conditions?

Quantitative analysis of At4g23960 protein expression requires rigorous methodological approaches:

Western Blot Quantification:

• Image Acquisition:

  • Capture images using a digital imaging system with linear dynamic range

  • Avoid overexposure which prevents accurate quantification

  • Include a standard curve of recombinant At4g23960 protein when possible

• Analysis Software:

  • Use ImageJ, Image Studio, or similar software for densitometry

  • Define regions of interest (ROIs) consistently across all bands

  • Subtract background signal from each measurement

• Normalization Strategies:

  • Normalize to housekeeping proteins or total protein stains

  • Calculate relative expression as: (Target protein signal / Loading control signal)

  • Present data as fold-change relative to control conditions

ELISA-Based Quantification:

• Standard Curve Generation:

  • Prepare a series of dilutions of recombinant At4g23960 protein

  • Plot absorbance values against known concentrations

  • Use four-parameter logistic regression for curve fitting

• Sample Quantification:

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

  • Run technical triplicates to assess measurement variability

  • Calculate protein concentration from the standard curve equation

Statistical Analysis:

• Perform appropriate statistical tests (t-test, ANOVA) to determine significance

• Report both fold-change and p-values

• Include error bars representing standard deviation or standard error

Similar quantitative approaches have been successfully used in antibody-based detection systems for other proteins, as demonstrated in the development of the A4 antibody system .

What are common sources of data inconsistency when using At4g23960 antibodies and how can they be addressed?

Several factors can contribute to data inconsistency when working with At4g23960 antibodies:

Sample Preparation Variables:

• Inconsistent extraction: Different extraction buffers or procedures can affect protein recovery

  • Solution: Standardize extraction protocol and buffer composition

  • Validation: Measure total protein recovery consistently

• Protein degradation: F-box proteins often have short half-lives

  • Solution: Include protease inhibitors and work at 4°C

  • Validation: Check for degradation products on Western blots

Antibody-Related Variables:

• Lot-to-lot variation: Different antibody batches may have different affinities

  • Solution: Purchase larger antibody lots when possible

  • Validation: Test new lots against old lots using the same samples

• Non-specific binding: Especially problematic in complex plant extracts

  • Solution: Optimize blocking conditions and antibody dilutions

  • Validation: Include knockout controls and pre-adsorption tests

Detection System Variables:

• Inconsistent transfer efficiency: Can affect Western blot results

  • Solution: Use stain-free gels or Ponceau S staining to verify transfer

  • Validation: Check membranes post-transfer for even protein distribution

• Signal saturation: Leads to underestimation of differences

  • Solution: Perform titration experiments to ensure linear detection range

  • Validation: Include a dilution series of a positive control sample

Experimental Design Strategies to Improve Consistency:

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

• Randomize sample processing order

• Process all samples for comparison in parallel

• Include internal reference samples across multiple experiments

How can researchers distinguish between specific and non-specific signals when using At4g23960 antibodies?

Distinguishing specific from non-specific signals is critical for accurate data interpretation:

Experimental Approaches:

• Knockout/Knockdown Controls:

  • Compare signal between wild-type and At4g23960 knockout plants

  • Specific signals should be absent or significantly reduced in knockouts

  • Example: Create a comparison table showing signal intensity in WT vs. knockout samples across different tissues

• Competition Assays:

  • Pre-incubate antibody with excess recombinant At4g23960 protein

  • Specific signals should be blocked by competition

  • Non-specific signals will remain unchanged

• Molecular Weight Verification:

  • Compare observed molecular weight with predicted size

  • Consider known post-translational modifications

  • Use high-resolution gels for better separation

• Multiple Antibodies Approach:

  • Use antibodies targeting different epitopes of At4g23960

  • Specific signals should be detected by multiple antibodies

  • Non-specific signals typically differ between antibodies

Analytical Methods to Distinguish Signal Types:

• Signal-to-noise ratio calculation:

  • Calculate as: (Specific band intensity - Background intensity) / Standard deviation of background

  • Higher ratios indicate more reliable detection

• Dose-response relationships:

  • Specific signals should show proportional changes with protein amount

  • Create titration curves with recombinant protein to establish linearity

• Signal pattern analysis across conditions:

  • Specific signals should show biologically plausible patterns

  • Non-specific signals often show random variation

How can computational approaches enhance the design and application of At4g23960 antibodies?

Recent advances in computational biology offer opportunities to enhance antibody design and application:

Computational Design Approaches:

• Epitope Prediction and Selection:

  • Use protein structure prediction to identify surface-exposed regions of At4g23960

  • Select epitopes with high antigenicity and low sequence similarity to other proteins

  • Computational tools can predict the most immunogenic regions

• Antibody Engineering:

  • Structure-based computational methods can optimize antibody-antigen interactions

  • Similar to approaches described in recent literature where computational pipelines incorporate physics- and AI-based methods for antibody design

  • Potential for improving specificity and affinity through in silico modeling

• Cross-Reactivity Prediction:

  • Sequence alignment and structural modeling can predict potential cross-reactivity

  • Helps in designing more specific antibodies by avoiding conserved regions

Implementation Strategy:

• Generate 3D models of At4g23960 using AlphaFold or similar tools

• Identify optimal epitopes through computational analysis

• Design antibodies with optimal binding characteristics

• Validate computationally designed antibodies experimentally

Similar computational approaches have proven successful in antibody design against viral targets, as demonstrated in recent research where machine learning-based antibody design approaches were combined with experimental validation to enable the design of high affinity and developable therapeutic antibodies .

What are emerging methodologies for improving At4g23960 antibody sensitivity and specificity?

Several cutting-edge approaches can enhance antibody performance:

Advanced Antibody Engineering:

• Single-chain variable fragments (scFvs):

  • Smaller antibody fragments with potentially better tissue penetration

  • Can be expressed recombinantly with consistent properties

• Nanobodies:

  • Single-domain antibody fragments derived from camelid antibodies

  • Excellent stability and ability to bind hidden epitopes

  • Potential for improved specificity against At4g23960

• Affinity maturation:

  • In vitro evolution of antibodies to enhance binding affinity

  • Can significantly improve detection sensitivity

Novel Detection Systems:

• Proximity ligation assay (PLA):

  • Combines antibody specificity with DNA amplification

  • Can dramatically increase sensitivity through signal amplification

  • Useful for detecting low-abundance At4g23960 protein

• Surface-enhanced Raman spectroscopy (SERS):

  • Similar to techniques used for the A4 antibody for neuraminidase detection

  • Can achieve ultrasensitive detection with minimal sample preparation

  • Potential for multiplexed detection of At4g23960 along with interaction partners

• Digital ELISA platforms:

  • Single-molecule detection capabilities

  • Can improve sensitivity by orders of magnitude over traditional ELISA

These emerging methodologies could significantly advance research into At4g23960 function and interactions, providing researchers with more powerful tools for studying this plant F-box protein.