At5g48990 Antibody

Basic Research Questions

• What is the At5g48990 gene and what protein does it encode?

The At5g48990 gene is found in Arabidopsis thaliana (Mouse-ear cress) and encodes a protein that belongs to a novel family of plant F-box proteins. This protein contains an amino-terminal F-box motif followed by four kelch repeats and a characteristic carboxy-terminal domain . As part of a 48-member gene family identified by 2001, this protein plays a potential role in the ubiquitin-dependent protein degradation pathway, which regulates numerous cellular processes in plants.

• What are the key structural features of the At5g48990 protein?

The At5g48990 protein contains three principal structural components:

• An N-terminal F-box motif that mediates protein-protein interactions, particularly with ASK1 proteins

• Four kelch repeats that form a potential protein-protein interaction domain, predicted by molecular modeling to form a β-propeller structure similar to the galactose oxidase crystal structure

• A characteristic carboxy-terminal domain with regulatory functions

These structural elements enable the protein to recruit specific target proteins for ubiquitination within cellular degradation pathways.

• What applications is the At5g48990 antibody validated for?

Based on available product information, the polyclonal At5g48990 antibody has been validated for:

• ELISA (Enzyme-Linked Immunosorbent Assay)

• Western Blotting (WB) with a recommended dilution of 1:1000

The antibody is typically raised in rabbit against recombinant Arabidopsis thaliana At5g48990 protein as the immunogen, and is antigen-affinity purified to ensure specificity .

Advanced Research Questions

• How has the functionality of the At5g48990 F-box motif been experimentally verified?

The F-box motif of the At5g48990 gene product has been experimentally verified through biochemical analysis demonstrating its functional activity. Specifically, researchers have shown that this motif mediates the in vitro interaction between the At5g48990 protein and ASK1 (Arabidopsis SKP1-like 1) proteins . This interaction is characteristic of F-box proteins, which typically function by recruiting specific target proteins to the SCF (Skp1-Cullin-F-box) ubiquitin ligase complex for subsequent ubiquitination and degradation by the 26S proteasome.

• What is the significance of the kelch repeats in At5g48990 for protein function?

The kelch repeats in At5g48990 form a β-propeller structure based on molecular modeling according to the galactose oxidase crystal structure . This structural arrangement creates a specialized protein-protein interaction platform that likely determines substrate specificity by identifying and binding to specific target proteins for ubiquitination. The presence of these repeats distinguishes this family from other F-box proteins that utilize different protein-protein interaction modules such as WD-40 domains or leucine-rich repeats (LRRs), suggesting distinct substrate recognition mechanisms.

• How does the At5g48990 protein compare with other plant F-box proteins?

The At5g48990 protein belongs to a distinct subset of the larger F-box protein superfamily in plants. While most characterized F-box proteins contain either WD-40 domains or leucine-rich repeats (LRRs) for substrate binding, the At5g48990 protein and its 47 family members uniquely combine an F-box motif with kelch repeats . This structural arrangement likely confers different substrate specificity compared to other F-box proteins. The identification of this family significantly expanded the known diversity of plant F-box proteins and highlighted additional potential mechanisms for regulated protein degradation in plant cellular processes.

Methodological Applications

• What is the optimal protocol for using At5g48990 antibody in Western blotting?

For optimal Western blotting with At5g48990 antibody:

• Sample preparation: Extract proteins from Arabidopsis tissue using an appropriate lysis buffer containing protease inhibitors

• Protein separation: Separate 20-50 μg of protein by SDS-PAGE (10-12% gel)

• Transfer: Transfer proteins to a PVDF or nitrocellulose membrane using standard methods

• Blocking: Block with 5% non-fat milk or BSA in TBST buffer for 1 hour at room temperature

• Primary antibody: Dilute At5g48990 antibody 1:1000 in blocking buffer

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

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

• Secondary antibody: Incubate with HRP-conjugated anti-rabbit IgG at appropriate dilution (typically 1:5000-1:10000)

• Detection: Visualize using ECL detection reagents and appropriate imaging system

When interpreting results, the antibody should detect a band corresponding to the molecular weight of the At5g48990 protein.

• How can researchers design effective ELISA experiments using At5g48990 antibody?

For optimal ELISA experiments with At5g48990 antibody:

• Format selection: Choose between direct, indirect, sandwich, or competitive ELISA based on research questions

• For sandwich ELISA (recommended for highest specificity):

  • Coat microplate wells with a capture antibody against At5g48990

  • Block remaining binding sites with appropriate blocking buffer

  • Add samples containing At5g48990 protein

  • Add detection antibody (either At5g48990 antibody or another antibody recognizing a different epitope)

  • Add enzyme-conjugated secondary antibody

  • Add substrate and measure signal

• Optimization considerations:

  • Antibody concentrations: Test serial dilutions to determine optimal concentration

  • Incubation times and temperatures: Optimize for maximum sensitivity

  • Blocking agents: Test different agents (BSA, casein, non-fat milk) to minimize background

  • Sample preparation: Ensure proper lysis and protein extraction from plant tissues

When developing sandwich ELISAs, consider using a combination of monoclonal and polyclonal antibodies against different epitopes of At5g48990 for improved specificity .

• What controls should be included when working with At5g48990 antibody?

Essential controls for experiments using At5g48990 antibody include:

For genetic validation, comparing signals between wild-type plants and At5g48990 knockouts/knockdowns provides the most definitive control. The antibody should show robust signal in wild-type samples and reduced or absent signal in genetic knockout samples.

Troubleshooting and Data Analysis

• How can researchers troubleshoot non-specific binding with At5g48990 antibody?

When encountering non-specific binding with At5g48990 antibody:

• Optimize antibody dilution: Test dilutions from 1:500 to 1:5000 to determine optimal concentration

• Modify blocking conditions:

  • Increase blocking agent concentration (5-10% BSA or non-fat milk)

  • Extend blocking time (2-3 hours or overnight)

  • Try alternative blocking agents (casein, normal serum, commercial blockers)

• Increase washing stringency:

  • Add 0.1-0.5% Triton X-100 or Tween-20 to washing buffers

  • Increase number of washes (5-7 washes)

  • Extend wash time (10-15 minutes per wash)

• Optimize sample preparation:

  • Include additional protease inhibitors

  • Pre-clear lysates by centrifugation at higher speeds

  • Pre-absorb antibody with proteins from non-target species

The At5g48990 antibody is antigen affinity-purified , which should reduce non-specific binding compared to crude serum preparations, but optimization for your specific experimental system is still essential.

• How should researchers approach contradictory data when using At5g48990 antibody?

When encountering contradictory results:

• Verify antibody specificity:

  • Perform peptide competition assays

  • Test in confirmed knockout/knockdown lines

  • Compare results with different antibody lots

• Cross-validate with alternative methods:

  • Compare protein detection with mRNA expression (RT-PCR)

  • Use mass spectrometry to confirm protein identity

  • Employ epitope-tagged versions of At5g48990

• Systematically evaluate experimental variables:

  • Different extraction methods for various subcellular compartments

  • Different tissue types or developmental stages

  • Various stress or treatment conditions

• Consider biological context:

  • Potential post-translational modifications affecting antibody recognition

  • Protein degradation during sample preparation

  • Expression variations due to environmental conditions or circadian regulation

Maintaining detailed experimental records and consulting literature specific to F-box proteins in plants can help resolve contradictory findings.

• What approaches can validate At5g48990 antibody specificity in experimental systems?

To comprehensively validate antibody specificity:

• Genetic approach:

  • Test antibody in At5g48990 knockout or knockdown lines

  • Compare signal between wild-type and mutant plants under identical conditions

• Biochemical validation:

  • Perform peptide competition assays with the immunizing peptide

  • Pre-absorb antibody with recombinant At5g48990 protein

  • Confirm band size corresponds to predicted molecular weight

• Advanced validation techniques:

  • Immunoprecipitation followed by mass spectrometry identification

  • Comparison with epitope-tagged At5g48990 expression

  • Multiple antibody approach (different antibodies targeting different epitopes)

• Cross-reactivity assessment:

  • Test antibody against closely related proteins

  • Evaluate in different plant species to determine conservation

Documenting validation experiments thoroughly in laboratory records and publications ensures experimental reproducibility and builds confidence in research findings.

Experimental Design for Advanced Applications

• How can researchers design gene expression studies to correlate with At5g48990 protein levels?

For comprehensive correlation studies:

• Experimental design considerations:

  • Include multiple tissue types and developmental stages

  • Design time-course experiments for dynamic processes

  • Include appropriate environmental stress treatments

• mRNA quantification methods:

  • qRT-PCR with validated reference genes

  • RNA-Seq for genome-wide expression context

  • In situ hybridization for spatial expression patterns

• Protein quantification methods:

  • Quantitative Western blotting with At5g48990 antibody

  • Quantitative immunohistochemistry for spatial patterns

  • Selected reaction monitoring (SRM) mass spectrometry

• Data integration strategies:

  • Calculate correlation coefficients between mRNA and protein

  • Use statistical models accounting for temporal delays

  • Apply clustering methods to identify co-regulated genes

When interpreting results, remember that F-box proteins often show poor correlation between mRNA and protein levels due to rapid turnover in the ubiquitin pathway, requiring careful experimental design and analysis.

• How can researchers investigate At5g48990 protein-protein interactions?

To investigate At5g48990 protein interactions:

• In vitro methods:

  • Pull-down assays with recombinant At5g48990 protein

  • Far-Western blotting to detect interacting proteins

  • Surface plasmon resonance for interaction kinetics

• In vivo methods:

  • Co-immunoprecipitation with At5g48990 antibody

  • Yeast two-hybrid with At5g48990 as bait

  • Bimolecular fluorescence complementation in planta

• Proximity-based methods:

  • Proximity-dependent biotin identification (BioID)

  • Förster resonance energy transfer (FRET)

  • Cross-linking followed by mass spectrometry

• Specialized approaches for F-box proteins:

  • SCF complex reconstitution assays

  • Substrate trapping with proteasome inhibitors

  • Degron motif identification in potential substrates

Given that At5g48990's F-box motif has been shown to interact with ASK1 proteins , this provides an excellent positive control for interaction studies and a foundation for building a complete interaction network.

• What approaches can identify potential substrates of the At5g48990-containing SCF complex?

To identify potential substrates:

• Proteomics-based methods:

  • Quantitative proteomics comparing wild-type vs. At5g48990 knockout plants

  • Immunoprecipitation of At5g48990 followed by mass spectrometry

  • Ubiquitin remnant profiling to identify ubiquitinated proteins

• Genetic approaches:

  • Suppressor screens of At5g48990 mutant phenotypes

  • Synthetic lethality screens

  • Analysis of genetic interactions through double mutants

• Biochemical methods:

  • In vitro ubiquitination assays with reconstituted SCF complexes

  • Protein stability assays in wild-type vs. At5g48990 mutants

  • Pulse-chase experiments to measure protein half-lives

• Computational predictions:

  • Analysis of proteins with known degron motifs

  • Co-expression network analysis

  • Structural modeling of kelch repeats to predict binding interfaces

Investigating multiple candidate substrates in parallel can accelerate discovery of the physiological roles of At5g48990 in plant development and stress responses.