At4g09190 Antibody

What is the At4g09190 protein and why is it studied in plant research?

At4g09190 is a putative F-box protein in Arabidopsis thaliana consisting of 383 amino acids. The protein is encoded by the At4g09190 gene and is also referenced as accession number NP_192658.1 in protein databases . F-box proteins are important components of SCF (Skp1-Cullin-F-box) ubiquitin ligase complexes that regulate protein degradation through the ubiquitin-proteasome pathway. These proteins play crucial roles in various cellular processes including hormone signaling, developmental regulation, and stress responses in plants. Researchers study At4g09190 to understand its potential role in protein-protein interactions and signaling pathways in Arabidopsis, which serves as a model organism for plant molecular biology research.

What types of At4g09190 antibodies are currently available for researchers?

There are three main types of At4g09190 antibodies available, each targeting different regions of the protein:

• N-terminus antibodies (X-Q9M0Q9-N): These are combinations of mouse monoclonal antibodies against the N-terminal sequence of At4g09190.

• C-terminus antibodies (X-Q9M0Q9-C): These target the C-terminal region of the protein.

• Middle region antibodies (X-Q9M0Q9-M): These target non-terminus sequences in the middle of the protein .

Each antibody type is a combination of individual monoclonal antibodies (mAbs) developed against a panel of synthetic peptide antigens from the corresponding region. These combinatorial antibodies can be used directly in experiments or can be further deconvoluted into individual monoclonal antibodies if specific epitope targeting is required .

What are the standard applications for At4g09190 antibodies in plant molecular biology?

At4g09190 antibodies are primarily used in several key research applications:

The available antibodies have been tested for ELISA with titers of approximately 10,000, which corresponds to detection sensitivity of approximately 1 ng of target protein on Western blot . Researchers should optimize dilutions for their specific experimental conditions and sample types.

How should researchers design proper validation experiments for At4g09190 antibodies?

Proper validation of At4g09190 antibodies is critical for ensuring reliable experimental results. A comprehensive validation protocol should include:

• Specificity testing: Compare wild-type Arabidopsis with At4g09190 knockout or knockdown lines. The antibody should show reduced or absent signal in genetic backgrounds where the protein is not expressed.

• Cross-reactivity assessment: Test the antibody against closely related F-box proteins to ensure it doesn't cross-react with other proteins. This is particularly important given the large family of F-box proteins in Arabidopsis.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before performing Western blot or immunostaining. A specific antibody will show diminished signal when its binding sites are blocked by the peptide.

• Recombinant protein controls: Express and purify recombinant At4g09190 protein to use as a positive control in immunoblotting experiments.

• Epitope mapping: If using multiple antibodies targeting different regions of the protein, confirm that they recognize the same protein band of the expected molecular weight.

These validation steps are essential since At4g09190 is classified as "AbClass™ Hard" , indicating potential challenges in antibody development and application.

What are the critical considerations for optimizing immunoprecipitation protocols with At4g09190 antibodies?

Optimizing immunoprecipitation (IP) protocols for At4g09190 requires careful attention to several factors:

• Lysis buffer composition: For F-box proteins like At4g09190, use buffers containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, with protease inhibitors. Consider adding phosphatase inhibitors if studying phosphorylation states.

• Crosslinking considerations: For transient interactions, consider using formaldehyde (1% for 10 minutes) or DSP (dithiobis(succinimidyl propionate)) crosslinking before lysis to capture weak or dynamic protein-protein interactions.

• Antibody selection: For IP experiments, the epitope accessibility in the protein's native conformation is crucial. Based on the available antibodies, researchers should test all three region-specific antibodies (N-terminus, C-terminus, and M-terminus) to identify which best recognizes the native protein .

• Bead selection: Protein A/G beads work well for mouse monoclonal antibodies like those available for At4g09190. Pre-clear lysates with beads alone to reduce non-specific binding.

• Elution strategy: For mass spectrometry applications, consider peptide elution using the immunizing peptide rather than denaturing elution to maintain the integrity of interacting proteins.

• Validation of results: Confirm IP results with reciprocal IP of interacting partners and through alternative methods like proximity ligation assays.

How does epitope selection affect the specificity and utility of At4g09190 antibodies?

The epitope selection is a critical determinant of antibody specificity and experimental utility:

• N-terminal antibodies (X-Q9M0Q9-N): The N-terminus of At4g09190 contains the sequence "MRKIQRITQFSDDNNRSQREHIPLDLIVEIVSSL" , which includes part of the F-box domain. These antibodies may be particularly useful for detecting the full-length protein but might not recognize truncated forms missing the N-terminus.

• C-terminal antibodies (X-Q9M0Q9-C): Target the C-terminal region containing "MHGGIAVDDIRRLDEVGYDLMKDLTVIPNHIQI" . These antibodies are valuable when studying proteins where the N-terminus might be processed or masked by protein interactions.

• Middle region antibodies (X-Q9M0Q9-M): Target non-terminus sequences that may include functional domains. These are particularly valuable for studying protein functionality or for detection when termini are inaccessible.

What are the recommended protocols for Western blot analysis using At4g09190 antibodies?

For optimal Western blot results with At4g09190 antibodies, follow this detailed protocol:

• Sample preparation:

  • Extract total protein from Arabidopsis tissues using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitors.

  • Quantify protein concentration using Bradford or BCA assay.

  • Mix 20-50 μg of total protein with Laemmli buffer and heat at 95°C for 5 minutes.

• Gel electrophoresis:

  • Separate proteins on a 10-12% SDS-PAGE gel (At4g09190 is 383 amino acids with an expected size of approximately 42-45 kDa).

  • Include molecular weight markers and appropriate positive and negative controls.

• Transfer:

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

  • Verify transfer efficiency with Ponceau S staining.

• Blocking:

  • Block the membrane with 5% non-fat dry milk in TBST (TBS with 0.1% Tween-20) for 1 hour at room temperature.

• Primary antibody incubation:

  • Dilute At4g09190 antibody to 1:1000-1:5000 in blocking solution.

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

  • Based on the antibody ELISA titer of 10,000, detection sensitivity should be approximately 1 ng of target protein .

• Washing:

  • Wash membrane 3-4 times with TBST, 5-10 minutes each.

• Secondary antibody:

  • Incubate with HRP-conjugated anti-mouse IgG (since the available At4g09190 antibodies are mouse monoclonal ) at 1:5000-1:10000 dilution for 1 hour at room temperature.

• Detection:

  • Develop using enhanced chemiluminescence (ECL) reagent.

  • Exposure time should be optimized based on signal strength.

• Controls to include:

  • Positive control: Extract from tissues known to express At4g09190

  • Negative control: Extract from At4g09190 knockout/knockdown lines

  • Loading control: Antibody against a housekeeping protein like actin or GAPDH

How can researchers effectively use At4g09190 antibodies for protein localization studies?

For effective protein localization studies using At4g09190 antibodies, consider the following comprehensive approach:

• Tissue fixation and preparation:

  • Fix Arabidopsis tissues in 4% paraformaldehyde for 2-4 hours.

  • For better penetration, consider vacuum infiltration in fixative.

  • Wash in PBS and either embed in paraffin/resin for sectioning or proceed with whole-mount immunostaining.

• Antigen retrieval:

  • For fixed tissues, perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10-20 minutes to expose epitopes that might be masked during fixation.

• Permeabilization and blocking:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS for 15-30 minutes.

  • Block with 3-5% BSA or normal serum in PBS for 1-2 hours.

• Antibody incubation:

  • Test all three antibody types (N-terminal, C-terminal, and middle region) to determine which provides optimal results for localization studies.

  • Dilute primary antibody 1:100-1:500 in blocking solution.

  • Incubate samples overnight at 4°C.

• Washing and secondary antibody:

  • Wash 3-5 times with PBS containing 0.1% Tween-20.

  • Incubate with fluorophore-conjugated secondary antibody (anti-mouse IgG) at 1:200-1:500 dilution for 1-2 hours at room temperature.

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI.

  • For plant tissues, consider counterstaining cell walls with calcofluor white or propidium iodide.

  • Mount in anti-fade mounting medium.

• Confocal imaging:

  • Use appropriate excitation/emission settings for the fluorophore.

  • Perform z-stack imaging to capture localization throughout the cell.

• Controls:

  • Include peptide competition controls to verify antibody specificity.

  • Use At4g09190 knockout/knockdown plants as negative controls.

  • Include known subcellular markers for co-localization studies.

How should researchers quantify and statistically analyze Western blot data for At4g09190 protein?

Proper quantification and statistical analysis of Western blot data for At4g09190 requires rigorous methodology:

• Image acquisition:

  • Capture images within the linear dynamic range of the detection system.

  • Avoid pixel saturation which prevents accurate quantification.

  • Include a dilution series of samples to verify linearity of detection.

• Densitometric analysis:

  • Use software like ImageJ, Image Lab, or commercial alternatives.

  • Draw identical boxes around each band of interest and a nearby area for background subtraction.

  • Calculate the integrated density value (IDV) for each band.

  • Normalize At4g09190 band intensity to loading control (actin/GAPDH) by dividing At4g09190 IDV by control IDV.

• Biological and technical replicates:

  • Perform at least three biological replicates (independent plant samples).

  • Include multiple technical replicates for each biological sample.

• Statistical analysis:

  • For comparing two groups: Use Student's t-test if data is normally distributed.

  • For comparing multiple groups: Use one-way ANOVA followed by post-hoc tests (Tukey's or Bonferroni).

  • For non-normally distributed data: Use non-parametric tests like Mann-Whitney or Kruskal-Wallis.

• Data presentation:

  • Present data as mean ± standard deviation or standard error.

  • Include p-values to indicate statistical significance.

  • Show representative blot images alongside quantification graphs.

This approach ensures reliable quantification of At4g09190 protein levels, which is particularly important given the antibody's high sensitivity (detection of approximately 1 ng of target protein) .

How can researchers interpret potential post-translational modifications of At4g09190 detected by Western blotting?

Interpreting post-translational modifications (PTMs) of At4g09190 requires careful analysis:

• Band shift analysis:

  • The unmodified At4g09190 protein (383 amino acids) has an expected molecular weight of approximately 42-45 kDa.

  • Multiple bands or bands at unexpected sizes may indicate PTMs:

    • Higher MW bands may suggest ubiquitination, SUMOylation, or glycosylation

    • Phosphorylation typically causes small shifts of 1-5 kDa

    • Lower MW bands may indicate proteolytic processing

• Confirmation strategies:

  • Phosphorylation: Treat protein extracts with lambda phosphatase before Western blotting; disappearance of shifted bands confirms phosphorylation.

  • Ubiquitination: Co-immunoprecipitate At4g09190 and probe with anti-ubiquitin antibodies.

  • Glycosylation: Treat samples with glycosidases (PNGase F for N-linked or O-glycosidase for O-linked glycans).

• Multiple antibody approach:

  • Compare results from N-terminal, C-terminal, and middle region antibodies .

  • Different banding patterns between antibody types may indicate region-specific modifications or processing.

• Mass spectrometry verification:

  • For definitive identification of PTMs, immunoprecipitate At4g09190 and analyze by LC-MS/MS.

  • This can identify specific modified residues and modification types.

• Biological significance assessment:

  • Compare PTM patterns across different tissues, developmental stages, and stress conditions.

  • Correlate changes in PTM patterns with known F-box protein regulatory mechanisms.

What are the common issues when using At4g09190 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with At4g09190 antibodies:

Since At4g09190 antibodies are classified as "AbClass™ Hard" , these issues may be more pronounced, requiring more extensive optimization compared to antibodies against abundant proteins.

How should researchers optimize antibody concentrations for different applications?

Optimization of At4g09190 antibody concentrations is critical for different applications:

• Western blotting titration:

  • Start with a dilution series: 1:500, 1:1000, 1:2000, 1:5000

  • Based on the ELISA titer of 10,000 , expect optimal dilutions around 1:1000-1:2000

  • Evaluate signal-to-noise ratio at each concentration

  • The optimal dilution provides clear specific bands with minimal background

• Immunofluorescence optimization:

  • Begin with higher concentrations: 1:50, 1:100, 1:200, 1:500

  • Include negative controls at each concentration

  • The optimal dilution should show clear subcellular localization with minimal background fluorescence

• Immunoprecipitation optimization:

  • Test antibody amounts: 1-5 μg per reaction

  • Evaluate IP efficiency by Western blotting both input and IP samples

  • Compare the three available antibody types (N, C, and M-terminal) for IP efficiency

• Antibody validation metrics:

  • Signal-to-noise ratio should be >3:1

  • Target band intensity should correlate with protein loading

  • Specificity should be confirmed with appropriate controls

• Optimization documentation:

  • Record detailed conditions for successful experiments

  • Note lot numbers of effective antibodies

  • Document tissue/extraction methods that yield best results

This systematic optimization is essential given that all three available antibodies (X-Q9M0Q9-N, X-Q9M0Q9-C, and X-Q9M0Q9-M) are combinations of multiple monoclonal antibodies , which may exhibit different optimal working conditions.

How can At4g09190 antibodies be used in protein interaction studies to characterize F-box protein complexes?

F-box proteins like At4g09190 typically function in protein complexes, particularly SCF ubiquitin ligase complexes. Here's a comprehensive approach to studying these interactions:

• Co-immunoprecipitation (Co-IP):

  • Use At4g09190 antibodies to pull down the protein complex from plant extracts.

  • Analyze co-precipitated proteins by Western blotting using antibodies against known SCF components (ASK/Skp1, Cullin1, RBX1).

  • For unknown interactors, analyze immunoprecipitates by mass spectrometry.

  • Consider using all three antibody types (N, C, and M-terminal) as epitope accessibility may differ in protein complexes.

• Proximity-dependent labeling:

  • Generate fusion proteins of At4g09190 with BioID or TurboID.

  • Express in Arabidopsis to biotinylate proximal proteins.

  • Purify biotinylated proteins using streptavidin.

  • Identify by mass spectrometry.

  • Validate interactions using At4g09190 antibodies in reverse Co-IP.

• Yeast two-hybrid validation:

  • Screen for interactors using Y2H.

  • Validate in planta interactions using split-GFP or FRET.

  • Confirm native interactions using At4g09190 antibodies.

• Immunofluorescence co-localization:

  • Perform dual immunostaining with At4g09190 antibodies and antibodies against putative interactors.

  • Analyze co-localization using confocal microscopy.

  • Calculate Pearson's correlation coefficient to quantify co-localization.

• In vitro binding assays:

  • Use purified recombinant At4g09190 protein.

  • Perform pull-down assays with putative interactors.

  • Verify interactions by immunoblotting with At4g09190 antibodies.

These approaches can help characterize both stable interactions (core SCF components) and transient interactions (substrates targeted for ubiquitination).