At4g34170 Antibody

What is the target protein of At4g34170 antibody and its biological function?

The At4g34170 antibody targets the putative F-box/kelch-repeat protein in Arabidopsis thaliana, encoded by the At4g34170 gene. This protein consists of 293 amino acids and functions as part of the SCF (Skp1-Cullin-F-box) complex involved in protein ubiquitination and subsequent degradation . The protein contains characteristic F-box domains that mediate protein-protein interactions and kelch-repeat motifs that are involved in substrate recognition.

When designing experiments with this antibody, researchers should consider that F-box proteins typically exhibit substrate specificity, making this antibody valuable for investigating specific protein degradation pathways in plants. The methodological approach should account for the protein's role in the ubiquitin-proteasome pathway when developing experimental protocols and analyzing results.

What techniques can be used to validate At4g34170 antibody specificity?

Validating antibody specificity for At4g34170 requires multiple complementary approaches:

• Western blot analysis comparing wild-type plants with At4g34170 knockout mutants

• Peptide competition assays using the immunizing peptide

• Immunoprecipitation followed by mass spectrometry confirmation

• Side-by-side comparison of different terminal-targeting antibodies (N, C, and M terminus)

The ELISA titer for commercially available At4g34170 antibodies is approximately 10,000, corresponding to detection sensitivity of around 1 ng of target protein on Western blots . When planning validation experiments, researchers should perform antibody dilution series (1:1000-1:10,000) to determine optimal working concentrations for their specific application and plant tissue type.

How should At4g34170 antibodies be stored to maintain reactivity?

For optimal preservation of At4g34170 antibody activity, storage conditions should be strictly followed:

• Store lyophilized antibody at -20°C until reconstitution

• After reconstitution with the recommended buffer (typically PBS pH 7.4), create small aliquots to avoid repeated freeze-thaw cycles

• Do not store At4g34170 antibodies at 4°C for extended periods

• Always briefly centrifuge tubes before opening to collect any material adhering to caps or tube walls

This methodological approach to storage is critical because monoclonal antibodies are susceptible to degradation pathways such as deamidation of asparagine residues, which can alter binding specificity and affinity . Proper storage prevents aggregation and maintains the structural integrity of the antibody's antigen-binding sites.

What are the optimal protocols for using At4g34170 antibody in different experimental techniques?

Different experimental applications require specific methodological considerations:

When performing Western blot analysis, researchers should be aware that the At4g34170 protein migrates at approximately 32-35 kDa despite its calculated mass of 33.2 kDa. This slight discrepancy is due to post-translational modifications and the presence of charged residues affecting electrophoretic mobility . For tissue-specific expression studies, it's crucial to optimize protein extraction buffers to prevent degradation of F-box proteins, which can have relatively short half-lives due to auto-ubiquitination.

How can researchers troubleshoot non-specific binding issues with At4g34170 antibody?

When encountering non-specific binding with At4g34170 antibody, implement this systematic troubleshooting approach:

• Increase blocking stringency using 5% non-fat milk with 0.1% Tween-20 in TBS

• Perform antibody pre-adsorption against plant extract from At4g34170 knockout mutants

• Compare reactivity patterns across different antibody combinations (N, C, and M terminus targeting)

• Optimize antigen retrieval methods for fixed tissues to improve epitope accessibility

The available antibody combinations against At4g34170 include separately targeting the N-terminus, C-terminus, and middle (non-terminus) regions of the protein . Each combination contains multiple monoclonal antibodies against synthetic peptide antigens from the corresponding region. This design allows researchers to select the most appropriate antibody combination based on experimental needs and potential protein interactions that might mask specific epitopes.

What approaches can detect post-translational modifications of At4g34170 using specific antibodies?

Detecting post-translational modifications (PTMs) of At4g34170 requires specialized methodological approaches:

• Compare migration patterns on Phos-tag gels to detect phosphorylation states

• Use deubiquitinating enzymes before immunoblotting to assess ubiquitination

• Employ mass spectrometry following immunoprecipitation to identify specific PTM sites

• Generate modification-specific antibodies for key regulatory sites

As an F-box protein, At4g34170 likely undergoes dynamic regulation through phosphorylation and auto-ubiquitination. Research indicates that F-box proteins can be regulated by phosphorylation at conserved residues, affecting their binding to both the SCF complex and their substrates. When designing experiments to study these modifications, researchers should consider extracting proteins under denaturing conditions with phosphatase and protease inhibitors to preserve the native modification state.

How can researchers analyze protein-protein interactions involving At4g34170 using antibody-based methods?

For studying protein-protein interactions involving At4g34170, several antibody-dependent methodologies can be employed:

• Co-immunoprecipitation followed by mass spectrometry

• Proximity ligation assay (PLA) for in situ detection of interactions

• Bimolecular fluorescence complementation (BiFC) validated with antibody detection

• Chromatin immunoprecipitation (ChIP) for DNA-protein interaction studies

When designing co-immunoprecipitation experiments, researchers should consider using reversible crosslinking agents to stabilize transient interactions, particularly since F-box proteins often have dynamic and substrate-dependent interaction profiles. The antibody combinations targeting different regions of At4g34170 can be strategically selected to avoid interference with specific protein-protein interaction domains .

How do different plant extraction buffers affect At4g34170 antibody performance?

The choice of extraction buffer significantly impacts At4g34170 antibody performance:

When extracting F-box proteins like At4g34170, researchers should be particularly careful about potential rapid degradation through the ubiquitin-proteasome pathway. Adding the proteasome inhibitor MG132 to extraction buffers can significantly improve protein yield and detection sensitivity. Additionally, performing extractions at 4°C and processing samples rapidly helps maintain protein integrity and antibody epitope recognition.

What are the differences in epitope accessibility when using different fixation methods for immunolocalization?

Different fixation methods significantly affect epitope accessibility for At4g34170 immunolocalization:

• Paraformaldehyde (4%): Preserves cellular architecture while maintaining good epitope accessibility; recommended for most applications

• Glutaraldehyde (0.1-0.5%): Stronger fixation but may mask epitopes; requires optimization for antigen retrieval

• Methanol: Preserves protein antigens but disrupts membrane structures; may alter subcellular localization interpretation

• Acetone: Good for preserving protein antigens but poor morphological preservation

When performing immunolocalization studies with At4g34170 antibody, researchers should optimize fixation protocols based on the specific plant tissue and subcellular compartment being investigated. For studying nuclear or cytosolic localization of At4g34170, paraformaldehyde fixation followed by permeabilization with 0.1% Triton X-100 typically provides the best balance between structural preservation and antibody accessibility.

How can researchers quantify At4g34170 protein levels accurately using antibody-based assays?

For accurate quantification of At4g34170 protein levels, several methodological considerations are essential:

• Include recombinant At4g34170 protein standards at known concentrations

• Employ fluorescent secondary antibodies for wider linear dynamic range

• Use image analysis software with background subtraction and normalization to loading controls

• Validate Western blot quantification with ELISA or protein mass spectrometry

When designing quantitative experiments, researchers should be aware that At4g34170, like many F-box proteins, may exhibit tissue-specific expression patterns and developmental regulation. The high sensitivity of available antibody combinations (detection limit ~1 ng) makes them suitable for detecting even low abundance protein in specialized tissues or under specific environmental conditions.

Can At4g34170 antibody be used for cross-species detection of homologous proteins?

When considering cross-species applications of the At4g34170 antibody, researchers should evaluate sequence conservation:

• Perform sequence alignment of the immunogenic peptide regions across species

• Test antibody reactivity on protein extracts from related plant species

• Validate specificity using recombinant proteins or knockout lines where available

• Consider epitope conservation when selecting between N, C, or M terminus-targeting antibodies

The current At4g34170 antibodies were developed against synthetic peptides representing different regions of the Arabidopsis thaliana protein . While not specifically tested for cross-reactivity, these antibodies may detect homologous F-box/kelch-repeat proteins in closely related species within the Brassicaceae family. For more distant species, researchers should perform careful validation experiments and consider developing species-specific antibodies if high specificity is required.

How do different At4g34170 antibody combinations compare in terms of specificity and sensitivity?

The three available antibody combinations for At4g34170 offer different performance characteristics:

Each antibody combination contains multiple monoclonal antibodies targeting different epitopes within the specified region . This design improves detection reliability but requires careful validation to ensure specificity. When selecting between these options, researchers should consider the biological question being addressed—for example, using C-terminal antibodies when studying proteins potentially subject to N-terminal processing.