At1g49990 Antibody

Basic Research Questions

What is At1g49990 and what cellular functions does it perform?

At1g49990 encodes an F-box family protein in Arabidopsis thaliana that functions as a substrate recognition component within the SKP1-cullin 1-F-box (SCF) E3 ubiquitin ligase complex. The protein contains an F-box domain at its N-terminus and leucine-rich repeats (LRRs) that facilitate specific substrate recognition . F-box proteins like At1g49990 play critical roles in protein ubiquitination by binding target substrates and mediating their polyubiquitination, which typically leads to 26S proteasomal degradation (K11/K48 linkages) or altered protein function/localization (K63 linkages) .

The protein has been computationally described as containing cyclin-like and Skp2-like F-box domains (InterPro domains IPR001810 and IPR022364) . Its subcellular localization is predominantly cytosolic, with a SUBAcon consensus score of 0.573, suggesting it functions primarily in cytosolic protein degradation pathways .

What detection methods are available for studying At1g49990 expression?

Several complementary approaches can be employed to study At1g49990:

• Antibody-based detection: Custom antibodies against At1g49990 (e.g., CSB-PA881771XA01DOA) can be used for western blotting, immunoprecipitation, and immunolocalization . For optimal results, use fresh plant tissue extracts prepared with buffer containing protease inhibitors and reducing agents.

• Transcript analysis: RT-PCR and qPCR can detect At1g49990 mRNA using gene-specific primers. This approach requires:

  • High-quality RNA extraction (RIN > 8)

  • DNase treatment to remove genomic DNA

  • Validated reference genes for normalization

  • Thermal cycling on systems like Bio-Rad T100

• Genetic tools: T-DNA insertion lines (SALK_078881) and amiRNA knockdown constructs (CSHL_019807) targeting At1g49990 are available for functional studies .

• Protein tagging: Creating transgenic lines with epitope-tagged versions (FLAG, GFP, etc.) of At1g49990 enables detection without specific antibodies.

What are the key structural and biochemical properties of the At1g49990 protein?

At1g49990 has the following key properties that should be considered when designing experiments:

The protein sequence contains multiple domains that may affect antibody binding, including the F-box domain (cyclin-like) and Skp2-like F-box domain . When designing experiments, consider that these domains may interact with other proteins, potentially masking epitopes in native conditions.

Advanced Research Applications

How can I optimize immunoprecipitation protocols using At1g49990 antibodies?

For successful immunoprecipitation of At1g49990 and its interacting partners:

• Crosslinking considerations: Since At1g49990 functions in a complex (SCF E3 ligase), using reversible crosslinkers like DSP (dithiobis[succinimidyl propionate]) at 1-2 mM can stabilize transient interactions.

• Buffer optimization:

  • Base buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40

  • Critical additives: 10 mM MG132 (proteasome inhibitor), 10 mM N-ethylmaleimide (deubiquitinase inhibitor)

  • Protease inhibitor cocktail with specific inhibitors for plant proteases

• Antibody coupling:

  • Direct coupling to magnetic beads improves recovery of native complexes

  • Use 5-10 μg antibody per 1 mg total protein extract

  • Control IP with IgG from the same species as the At1g49990 antibody

• Validation methods:

  • Use SALK_078881 T-DNA mutant extracts as negative controls

  • Perform reciprocal IP with antibodies against known SCF components

  • Validate interactions with both western blot and mass spectrometry

Since F-box proteins like At1g49990 form part of larger SCF complexes with cullin-1 and SKP1, co-IP experiments should be designed to detect these interactions. Research on related F-box proteins shows that the F-box domain interacts with SKP1 while the leucine-rich repeats typically interact with substrates .

What are the recommended approaches for studying At1g49990's role in protein degradation?

To investigate At1g49990's function in protein degradation pathways:

• Substrate identification:

  • Perform IP-MS with At1g49990 antibodies under different conditions

  • Use cycloheximide chase assays with wild-type and fbxl-5 mutants

  • Apply proximity labeling techniques (BioID or TurboID fused to At1g49990)

• Ubiquitination assays:

  • In vitro: Reconstitute SCF^At1g49990^ complex with recombinant proteins

  • In vivo: Express HA-tagged ubiquitin and immunoprecipitate with At1g49990 antibody

  • Detection: Use anti-ubiquitin western blots to visualize polyubiquitinated substrates

• Proteasome dependency:

  • Treat samples with MG132 (10-25 μM) for 3-6 hours

  • Compare protein levels with/without proteasome inhibition

  • Analyze ubiquitination patterns of putative substrates

• Functional validation:

  • Create artificial substrates with degrons recognized by At1g49990

  • Use fluorescent timers to measure protein half-life changes

  • Compare substrate stability in wild-type vs. SALK_078881 mutant backgrounds

Similar approaches have been successfully used to study other F-box proteins like FBXL-5 in C. elegans, which was identified as a negative regulator of vitellogenin lipoproteins . This suggests At1g49990 may similarly regulate specific developmental or metabolic proteins in Arabidopsis.

How can I use At1g49990 antibodies in combination with genetic approaches?

Integrating antibody-based detection with genetic tools provides powerful insights:

• Complementary knockout/knockdown strategies:

  • T-DNA insertion mutant (SALK_078881) for complete gene knockout

  • amiRNA construct (CSHL_019807) for tissue-specific knockdown

  • CRISPR/Cas9 for generating precise mutations in functional domains

• Experimental design for genetic verification:

  • Use heterozygous plants from SALK_078881 line (kanamycin selection)

  • Confirm insertion by PCR genotyping

  • Verify protein absence via western blot with At1g49990 antibody

  • Perform complementation with wild-type At1g49990 to rescue phenotypes

• Tissue-specific analyses:

  • Drive amiRNA expression with tissue-specific promoters

  • Validate knockdown efficiency by western blot in isolated tissues

  • Compare protein levels between different tissues using calibrated western blots

• Interaction studies in genetic backgrounds:

  • Cross mutant lines with tagged versions of potential interactors

  • Perform IP with At1g49990 antibodies in different genetic backgrounds

  • Quantify differences in interaction partners using quantitative MS

These approaches have proven effective for studying other F-box proteins, as demonstrated in work on CAND1, which is required for pollen viability in Arabidopsis and functions in the dynamic assembly of SCF complexes .

What considerations are important when using At1g49990 antibodies for tissue-specific expression analysis?

When examining tissue-specific expression patterns:

• Tissue preparation protocols:

  • For reproductive tissues: Use modified extraction buffer with 1% PVP-40 to remove phenolics

  • For high-resolution imaging: Consider ethanol:acetic acid (3:1) fixation followed by paraffin embedding

  • For maintained protein interactions: Use mild crosslinking (1% formaldehyde, 10 min)

• Quantitative comparison between tissues:

  • Use identical protein amounts (15-30 μg) from different tissues

  • Include spike-in controls with known concentrations

  • Use tissue-specific housekeeping proteins for normalization

  • Apply fluorescent western blot for accurate quantification

• Immunohistochemistry optimization:

  • Fixation: 4% paraformaldehyde for 2-4 hours (depending on tissue)

  • Antigen retrieval: Citrate buffer (pH 6.0) at 95°C for 10-15 minutes

  • Blocking: 5% BSA in PBS with 0.1% Triton X-100

  • Primary antibody dilution: Start at 1:100-1:500 and optimize

  • Detection: Fluorescent secondary antibodies for co-localization studies

• Controls and validation:

  • Pre-immune serum at same concentration as primary antibody

  • Peptide competition assay (pre-incubate antibody with immunizing peptide)

  • Parallel staining of T-DNA mutant tissue (SALK_078881)

Research on related F-box proteins suggests that expression may vary significantly across tissues and developmental stages, with some showing higher expression in reproductive tissues as observed for F-box proteins involved in pollen viability .

How does the F-box domain of At1g49990 influence antibody selection and experimental design?

The F-box domain creates specific considerations for antibody design and experiment planning:

• Epitope selection strategy:

  • Avoid using the F-box domain (N-terminal region) as an epitope since it interacts with SKP1

  • Target unique regions within the leucine-rich repeats that aren't conserved in related proteins

  • C-terminal peptides may offer better specificity than N-terminal regions

  • Consider raising antibodies against multiple regions for validation

• Structural implications for experiments:

  • In native conditions, the F-box domain may be occluded in the SCF complex

  • Denaturing conditions may be required for consistent detection in western blots

  • Epitope masking may occur during protein-protein interactions

• Cross-reactivity assessment:

  • Test against recombinant proteins of related F-box family members

  • Perform western blots on tissue from knockout lines of related F-box genes

  • Use peptide competition assays with related and unrelated peptide sequences

• Functional domain preservation:

  • For tagged protein constructs, avoid N-terminal tags that might disrupt F-box function

  • C-terminal tags are preferable for maintaining native interactions

  • Validate functionality of tagged proteins by complementation tests

Similar considerations have been important when studying antibodies against other domains, as demonstrated in research on PD-1 specific antibodies where epitope mapping revealed that different antibody clones had varying abilities to detect PD-1 depending on its binding state .

Troubleshooting and Methodology Refinement

What are the most common issues when using At1g49990 antibodies and how can they be resolved?

When working with At1g49990 antibodies, researchers may encounter several challenges:

• Low signal intensity:

  • Cause: Low protein abundance or poor extraction

  • Solution: Enrich samples via immunoprecipitation before western blot

  • Method: Use 500-1000 μg total protein for IP, then load entire eluate

• Multiple bands on western blots:

  • Cause: Post-translational modifications or degradation products

  • Solution: Use freshly prepared samples with multiple protease inhibitors

  • Validation: Compare pattern in wild-type vs. SALK_078881 mutant

• Variable results between experiments:

  • Cause: Different growth conditions affecting protein expression

  • Solution: Standardize growth conditions (light, temperature, humidity)

  • Control: Include biological replicates and standardized positive control

• Background in immunolocalization:

  • Cause: Nonspecific binding or autofluorescence

  • Solution: Increase blocking time (overnight at 4°C) with 5% BSA

  • Alternative: Try different fixatives (ethanol:acetic acid vs. paraformaldehyde)

• Failed co-immunoprecipitation:

  • Cause: Harsh buffer conditions disrupting interactions

  • Solution: Use milder detergents (0.1% NP-40 instead of 1% Triton X-100)

  • Improvement: Add stabilizing agents like 10% glycerol to buffers

Each of these issues can be systematically addressed through protocol optimization and proper controls, similar to approaches used in other plant protein studies .

How can I adapt extraction protocols to optimize At1g49990 detection in different plant tissues?

Different plant tissues require specific extraction modifications:

• Leaf tissue protocol:

  • Buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% NP-40

  • Critical additions: 1 mM DTT, 1 mM PMSF, plant protease inhibitor cocktail

  • Physical disruption: Grind in liquid nitrogen, then thaw in buffer

  • Clarification: 15,000 × g centrifugation for 15 minutes at 4°C

• Root tissue modifications:

  • Increase detergent to 0.5% NP-40

  • Add 1% PVP-40 to remove phenolic compounds

  • Include 5 mM EDTA to inhibit metal-dependent proteases

  • Perform additional clarification step (20,000 × g for 20 minutes)

• Flower/reproductive tissue adaptations:

  • Add 2% PVPP to binding buffer to remove secondary metabolites

  • Increase DTT to 5 mM

  • Use shorter extraction time (minimize exposure to air)

  • Consider phase separation with TCA/acetone if phenolics remain problematic

• Seed tissue approach:

  • Pre-soak seeds in extraction buffer for 30 minutes

  • Use mechanical disruption (bead-beating) after liquid nitrogen grinding

  • Filter lysate through Miracloth before centrifugation

  • Perform protein precipitation with TCA/acetone if lipids interfere

These tissue-specific modifications address the unique biochemical composition of each tissue type, improving protein yield and maintaining native protein state, similar to approaches used in other plant molecular biology studies .

What are the critical considerations for comparing At1g49990 protein levels across experimental conditions?

For accurate quantitative comparisons:

• Standardized sample preparation:

  • Harvest tissues at the same time of day (protein levels may vary diurnally)

  • Use identical growth conditions (light intensity, photoperiod, temperature)

  • Process all samples simultaneously with the same reagent batches

  • Prepare multiple biological replicates (minimum n=3)

• Loading controls selection:

  • Use multiple loading controls (e.g., actin, tubulin, and GAPDH)

  • Validate stability of loading controls under your experimental conditions

  • Consider using total protein staining (Ponceau S or SYPRO Ruby) as alternative

• Quantification methods:

  • Use fluorescent secondary antibodies for linear dynamic range

  • Establish standard curves with recombinant protein if absolute quantification is needed

  • Perform normalization to multiple reference proteins

  • Use software that measures integrated density rather than peak intensity

• Statistical analysis:

  • Apply appropriate statistical tests based on data distribution

  • Use ANOVA for multiple condition comparisons followed by post-hoc tests

  • Report fold changes with confidence intervals

  • Consider power analysis to determine minimum sample size needed

These approaches ensure reliable quantitative comparisons between experimental conditions and have been validated in studies of other plant proteins .

Emerging Research Directions

How might At1g49990 be involved in plant stress responses based on its F-box protein classification?

Based on studies of related F-box proteins and functional domain analysis:

• Potential stress-related functions:

  • F-box proteins often mediate degradation of transcription factors that regulate stress responses

  • Leucine-rich repeats in At1g49990 suggest it may target phosphorylated substrates

  • Its cytosolic localization indicates it likely targets non-membrane proteins

• Experimental approaches to investigate stress involvement:

  • Compare At1g49990 protein levels across various abiotic stresses (drought, cold, salt)

  • Analyze phenotypes of SALK_078881 mutants under stress conditions

  • Perform RNA-seq of mutants vs. wild-type under normal and stress conditions

  • Identify potential substrates that accumulate in mutants during stress

• Protein modification during stress:

  • Monitor At1g49990 phosphorylation state during stress using Phos-tag gels

  • Examine protein stability under stress using cycloheximide chase assays

  • Test if stress conditions alter At1g49990 interaction partners

Research on related F-box proteins like FBXL-5 in C. elegans shows they can regulate critical metabolic processes like vitellogenesis and lipid metabolism , suggesting At1g49990 might similarly regulate metabolic adaptations to stress in Arabidopsis.

What techniques can be used to identify the specific substrates targeted by At1g49990?

To identify the specific proteins targeted by At1g49990 for ubiquitination:

• Proximity-based approaches:

  • BioID: Fuse At1g49990 to a promiscuous biotin ligase (BirA*)

  • TurboID: Use improved biotin ligase for faster labeling

  • APEX2: Proximity-based biotinylation with shorter labeling time

  • Experimental design: Express fusion protein, add biotin, purify biotinylated proteins, analyze by MS

• Differential proteomics:

  • Compare proteomes of wild-type vs. SALK_078881 mutant plants

  • Focus on proteins that accumulate in the mutant

  • Filter candidates by ubiquitination status (use ubiquitin remnant profiling)

  • Validate with in vitro ubiquitination assays

• Yeast two-hybrid screens:

  • Use leucine-rich repeat region as bait (exclude F-box domain)

  • Screen against Arabidopsis cDNA library

  • Validate interactions in planta using split-luciferase or FRET

  • Test if interactions are enhanced in proteasome-inhibited conditions

• In vitro binding assays:

  • Express recombinant At1g49990 protein domains

  • Perform protein array screening

  • Validate with pull-down assays using plant extracts

  • Test if phosphorylation affects binding (common for F-box substrates)

Such approaches have successfully identified substrates for other plant F-box proteins, revealing their roles in hormone signaling, development, and stress responses .