At3g04660 Antibody

What is the AT3G04660 protein in Arabidopsis thaliana and why would I need an antibody against it?

AT3G04660 encodes an F-box and associated interaction domains-containing protein in Arabidopsis thaliana. According to genomic data, this protein belongs to a family involved in protein-protein interactions and is potentially part of SCF (Skp, Cullin, F-box) complexes that participate in ubiquitin-mediated protein degradation pathways .

F-box proteins like AT3G04660 often function in various cellular processes including hormone signaling, development, and stress responses. Antibodies against this protein are valuable tools for:

• Detecting protein expression levels in different tissues or conditions

• Determining subcellular localization

• Studying protein-protein interactions

• Investigating protein degradation pathways

An antibody would be particularly useful if you're studying regulatory networks involving this F-box protein or investigating its role in specific developmental or stress response pathways.

What are the recommended methods for validating an AT3G04660 antibody's specificity?

Proper validation of antibody specificity is crucial for reliable research results. For AT3G04660 antibodies, implement these methodological approaches:

• Genetic controls: Test the antibody in wild-type versus knockout/knockdown lines (e.g., T-DNA insertion line SALK_007123 if available for this gene)

• Western blot analysis:

  • Expected molecular weight for AT3G04660 is approximately 57 kDa (similar to other F-box proteins)

  • Test antibody against recombinant AT3G04660 protein

  • Use appropriate negative controls (knockout lines) and positive controls (overexpression lines)

• Immunoprecipitation followed by mass spectrometry:

  • Similar to methods used for other Arabidopsis proteins like HDA9

  • Confirm that peptides matching AT3G04660 are enriched

• Blocking peptide assay:

  • Pre-incubate antibody with the immunizing peptide before immunodetection

  • Signal should be significantly reduced or eliminated

• Cross-reactivity testing:

  • Test against closely related F-box proteins to ensure specificity

  • Especially important given the large F-box protein family in Arabidopsis

What applications are suitable for AT3G04660 antibodies in basic plant research?

AT3G04660 antibodies can be utilized in multiple applications similar to other Arabidopsis protein antibodies:

• Western blotting:

  • Recommended dilution: 1:1000 (based on similar antibodies)

  • Sample preparation: Use a buffer optimized for membrane proteins, such as extraction buffers containing appropriate detergents

  • Allow detection of protein accumulation under different conditions or developmental stages

• Immunolocalization:

  • Dilution range: 1:25 to 1:500 (based on similar antibodies)

  • Can determine subcellular localization (likely nuclear/cytoplasmic based on function)

  • Fixation protocol should preserve protein epitopes while maintaining cellular architecture

• Immunoprecipitation:

  • Typically requires 2-5 μg of antibody per reaction

  • Useful for identifying interacting proteins

  • Can reveal protein complex formation under different conditions

• Chromatin Immunoprecipitation (ChIP):

  • If AT3G04660 has DNA-binding properties or associates with chromatin

  • Protocol similar to that used for other plant transcription factors

How should I store and handle AT3G04660 antibodies to maintain their activity?

Proper storage and handling of antibodies is critical for maintaining their specificity and sensitivity:

• Storage conditions:

  • Store lyophilized antibodies at -20°C

  • After reconstitution, make small aliquots to avoid repeated freeze-thaw cycles

  • Add preservatives like sodium azide (0.02%) for longer storage periods

• Reconstitution protocol:

  • Add sterile water as specified (typically 50 μl for lyophilized antibodies)

  • Allow complete dissolution before use (gentle mixing without vortexing)

• Stability considerations:

  • Antibody activity can decrease after multiple freeze-thaw cycles

  • Working dilutions should be prepared fresh

  • Monitor signal strength over time to detect potential degradation

• Quality control measures:

  • Include positive controls in each experiment

  • Run parallel experiments with previously tested antibody batches to ensure consistency

What approaches are recommended for detecting post-translational modifications of AT3G04660 using antibodies?

F-box proteins are commonly regulated by post-translational modifications (PTMs). For AT3G04660:

• Phosphorylation detection:

  • Use phospho-specific antibodies if available, or general phospho-detection after IP

  • Treat samples with phosphatase inhibitors during extraction

  • Compare phosphorylation status under different conditions (e.g., stress responses)

  • Utilize Phos-tag™ SDS-PAGE to separate phosphorylated forms

• Ubiquitination analysis:

  • Since F-box proteins often participate in ubiquitination pathways, they may be autoubiquitinated

  • IP using AT3G04660 antibody followed by Western blot with anti-ubiquitin

  • Include deubiquitinase inhibitors in extraction buffers

  • Consider using tagged ubiquitin expression systems for enhanced detection

• Other PTMs:

  • SUMOylation: IP followed by anti-SUMO Western blot

  • Glycosylation: Use specific glycosylation detection kits after IP

  • Acetylation: IP followed by anti-acetyl-lysine Western blot

• Mass spectrometry approach:

  • IP AT3G04660 and perform LC-MS/MS analysis

  • Look for mass shifts corresponding to known modifications

  • Use enrichment techniques specific for phosphopeptides or ubiquitinated peptides

Sample experimental design for phosphorylation analysis:

How can I use AT3G04660 antibodies to investigate protein degradation pathways in stress responses?

Since AT3G04660 is an F-box protein potentially involved in ubiquitin-mediated protein degradation:

• Stress-induced degradation analysis:

  • Apply various stresses (drought, salt, cold, pathogens)

  • Monitor AT3G04660 levels and those of potential substrates

  • Include proteasome inhibitors (MG132) to confirm proteasomal degradation

  • Compare wild-type vs mutant responses

• Substrate identification strategy:

  • Perform differential proteomics between wild-type and at3g04660 mutant

  • Look for proteins that accumulate in the mutant under specific conditions

  • Validate with targeted approaches using antibodies against candidate substrates

  • Confirm direct interaction by co-IP and in vitro ubiquitination assays

• Degradation dynamics visualization:

  • Use cycloheximide chase assays to monitor protein turnover rates

  • Pulse-chase experiments with metabolic labeling

  • Live-cell imaging with fluorescently tagged proteins

• SCF complex assembly analysis:

  • Co-IP to detect association with SCF components (ASK/Cullin/RBX)

  • Test for condition-dependent complex formation

  • Use mutants of SCF components to validate pathway

Based on studies of other F-box proteins in plants, a comprehensive approach might include:

What considerations should be made when developing an AT3G04660 antibody for studying circadian regulation?

Given that several Arabidopsis proteins like PRR5 are involved in circadian regulation , investigating AT3G04660's potential role requires specialized approaches:

• Antibody generation strategy:

  • Target epitopes that aren't subject to circadian-regulated modifications

  • Consider generating antibodies against multiple regions (N-terminal, C-terminal, middle region)

  • Validate antibody performance across all time points in a circadian cycle

• Time-course experimental design:

  • Sample collection every 4 hours across a 24-48 hour period

  • Maintain consistent growth conditions (light, temperature) before sampling

  • Include appropriate circadian mutants as controls

  • Use antibody to monitor protein abundance, modifications, and interactions

• Interaction with core clock components:

  • Co-IP experiments at different circadian time points

  • Test interactions with known clock components like PRR5, PRR7, and CCA1

  • Verify interactions using independent methods (yeast two-hybrid, BiFC)

• Protein modification across the circadian cycle:

  • Monitor phosphorylation status throughout the day/night cycle

  • Examine protein stability and degradation rates at different time points

  • Test effects of proteasome inhibitors on circadian rhythms

Methodology similar to that used for PRR5-ABI5 interaction studies :

ZT = Zeitgeber Time (hours after lights on)