At4g02650 Antibody

What is At4g02650 and what is its biological role in Arabidopsis thaliana?

At4g02650 (UniProt Number: Q8GX47) is a gene in Arabidopsis thaliana with the Entrez Gene ID 828212. While the complete biological function has not been fully characterized in the provided search results, it appears to be expressed during female gametophyte development in Arabidopsis. The gene product likely plays a role in reproductive development, as indicated by transcriptome analysis studies of the Arabidopsis female gametophyte . Researchers investigating this protein should consider its potential involvement in plant reproduction and development pathways when designing experiments.

What are the specifications of commercially available At4g02650 antibodies?

The commercially available At4g02650 antibody is a rabbit polyclonal antibody purified by Antigen Affinity techniques. The antibody is raised against recombinant Arabidopsis thaliana At4g02650 protein as the immunogen. The commercial preparation typically includes three components: 200μg antigens (used as positive control), 1ml pre-immune serum (used as negative control), and the rabbit polyclonal antibodies themselves . The antibody has been validated for ELISA and Western Blot (WB) applications and demonstrates reactivity with plant species .

How should I validate the specificity of the At4g02650 antibody in my experimental system?

For rigorous validation of At4g02650 antibody specificity, implement a multi-step approach. First, perform Western blot analysis using both the supplied positive control antigen (200μg) and the negative control pre-immune serum (1ml) that typically accompanies the antibody . Second, include appropriate biological controls in your experiments - ideally wild-type Arabidopsis tissue alongside At4g02650 knockout or knockdown lines if available.

For advanced validation, consider using transcriptome data from female gametophyte studies to identify tissues with differential At4g02650 expression levels . Comparing antibody reactivity between tissues with high and low expression provides additional evidence of specificity. When testing in plants other than Arabidopsis, perform preliminary cross-reactivity tests against protein extracts from multiple plant species at varying antibody dilutions to determine optimal conditions.

What are the optimal conditions for using At4g02650 antibody in Western blot applications?

For optimal Western blot results with At4g02650 antibody, consider the following methodological approach based on general antibody practices and the specific characteristics of this antibody:

• Sample preparation: Extract total protein from Arabidopsis tissues using a plant-specific extraction buffer containing protease inhibitors to prevent degradation. Based on gene expression data, female reproductive tissues may show higher expression levels .

• Gel electrophoresis: Separate 20-50μg of total protein on an SDS-PAGE gel (10-12% is typically suitable for most plant proteins).

• Transfer conditions: Transfer to a PVDF membrane at 100V for 60-90 minutes in standard transfer buffer.

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

• Primary antibody incubation: Dilute the At4g02650 antibody (start with 1:1000 and optimize as needed) in blocking buffer and incubate overnight at 4°C.

• Washing and secondary antibody: Wash with TBST and incubate with HRP-conjugated anti-rabbit secondary antibody (typically 1:5000 dilution) for 1 hour at room temperature.

• Detection: Develop using an enhanced chemiluminescence (ECL) system.

Always include the positive control antigen provided with the antibody to confirm detection functionality .

How can I use the At4g02650 antibody for immunolocalization studies in plant tissues?

For immunolocalization of At4g02650 in plant tissues, consider this methodological approach:

• Tissue fixation: Fix freshly harvested Arabidopsis tissues (preferably including female reproductive structures based on expression data ) in 4% paraformaldehyde for 2-4 hours at room temperature.

• Tissue processing: Dehydrate tissues through an ethanol series, clear with xylene, and embed in paraffin. Section tissues at 5-10μm thickness.

• Antigen retrieval: After deparaffinization, perform antigen retrieval using citrate buffer (pH 6.0) at 95°C for 10-20 minutes to expose epitopes that may have been masked during fixation.

• Blocking: Block with 2-5% BSA or normal goat serum in PBS with 0.1% Triton X-100 for 1 hour at room temperature.

• Primary antibody incubation: Dilute At4g02650 antibody (1:100 to 1:500, optimize as needed) in blocking solution and incubate sections overnight at 4°C.

• Secondary antibody: After washing, incubate with fluorophore-conjugated anti-rabbit secondary antibody (1:200 to 1:500) for 1-2 hours at room temperature.

• Counterstaining: Counterstain nuclei with DAPI (1μg/ml) for 5 minutes.

• Controls: Include negative controls using pre-immune serum and positive controls using tissues known to express At4g02650 based on transcriptome data .

• Analysis: Image using confocal microscopy with appropriate filter sets for your fluorophore.

What approaches can I use to study protein-protein interactions involving At4g02650?

To investigate protein-protein interactions involving At4g02650, consider these methodological strategies:

• Co-immunoprecipitation (Co-IP):

  • Prepare protein extracts from Arabidopsis tissues expressing At4g02650

  • Immobilize the At4g02650 antibody on protein A/G beads

  • Incubate with protein extracts to capture At4g02650 and its interacting partners

  • Wash extensively to remove non-specific interactions

  • Elute bound proteins and analyze by mass spectrometry

• Proximity-dependent biotin identification (BioID):

  • Generate transgenic plants expressing At4g02650 fused to a biotin ligase (BirA*)

  • The BirA* will biotinylate proteins in close proximity to At4g02650 in vivo

  • Purify biotinylated proteins using streptavidin beads

  • Identify interacting proteins by mass spectrometry

• Yeast two-hybrid screening:

  • Clone At4g02650 as bait in a yeast two-hybrid system

  • Screen against an Arabidopsis cDNA library

  • Validate positive interactions through secondary screens

These approaches can be particularly valuable for understanding the molecular function of At4g02650, especially given its potential role in female gametophyte development as suggested by transcriptome data .

How does At4g02650 expression compare across different developmental stages in Arabidopsis?

While comprehensive developmental expression data specifically for At4g02650 is limited in the provided search results, we can infer from the transcriptome analysis of female gametophytes that At4g02650 shows expression during reproductive development . The analysis of female gametophyte transcriptome in Arabidopsis compared heterozygous ovules (containing embryo sacs) with sporocyteless (spl) mutant ovules (lacking embryo sacs).

For researchers interested in At4g02650 expression patterns, it would be valuable to consult publicly available Arabidopsis expression databases like AtGenExpress or BAR eFP Browser, which provide tissue-specific and developmental stage-specific expression data across multiple experimental conditions. Additionally, researchers should consider performing RT-qPCR analysis across different tissues and developmental stages to generate a comprehensive expression profile for this gene before embarking on protein-level studies with the antibody.

What methodologies can I use to correlate At4g02650 protein abundance with its transcriptional activity?

To establish correlations between At4g02650 transcription and protein abundance:

• Paired analysis approach:

  • Collect Arabidopsis tissues from multiple developmental stages or treatment conditions

  • Split each sample into two portions

  • Extract RNA from one portion for RT-qPCR or RNA-seq analysis of At4g02650 transcript levels

  • Extract protein from the other portion for Western blot quantification using the At4g02650 antibody

  • Calculate correlation coefficients between transcript and protein levels

• Time-course studies:

  • Induce expression changes through developmental progression or environmental stimuli

  • Collect samples at regular intervals

  • Analyze both transcript and protein dynamics

  • Determine the temporal relationship between changes in transcript and protein levels

• Single-cell approaches:

  • For specialized tissues like female gametophytes , consider single-cell RNA-seq paired with immunofluorescence using the At4g02650 antibody

  • This can reveal cell-specific correlations between transcript and protein abundance

Include appropriate internal controls for both transcript analysis (reference genes) and protein quantification (loading controls) to ensure reliable correlations.

What are the common issues encountered with At4g02650 antibody and how can they be resolved?

When working with At4g02650 antibody, researchers may encounter several challenges:

• High background in Western blots:

  • Increase blocking time or blocking agent concentration (5-10% milk/BSA)

  • Increase washing duration and frequency (5 washes × 5 minutes with TBST)

  • Optimize primary antibody dilution (try 1:1000 to 1:5000 range)

  • Include 0.1-0.5% Tween-20 in antibody dilution buffer

• Weak or no signal:

  • Confirm protein expression using transcriptome data

  • Increase protein loading (50-100μg total protein)

  • Reduce antibody dilution (try 1:500)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Enhance detection with more sensitive ECL substrates

  • Use the positive control antigen provided with the antibody to verify antibody functionality

• Multiple bands or non-specific binding:

  • Increase washing stringency

  • Optimize antibody concentration

  • Pre-adsorb antibody with total protein extract from negative control tissues

  • Compare against the pre-immune serum control to identify non-specific bands

• Poor reproducibility:

  • Standardize protein extraction protocols

  • Aliquot antibody upon receipt to avoid freeze-thaw cycles

  • Document lot-to-lot variations of the antibody

  • Maintain consistent incubation times and temperatures

How should I optimize immunoprecipitation protocols when using At4g02650 antibody?

For optimal immunoprecipitation results with At4g02650 antibody:

• Buffer optimization:

  • Test different lysis buffers (RIPA, NP-40, or plant-specific buffers)

  • Include protease inhibitors and phosphatase inhibitors if studying phosphorylation

  • Adjust salt concentration (150-500mM NaCl) to reduce non-specific binding

• Antibody binding:

  • Determine optimal antibody-to-protein ratio (typically 2-5μg antibody per 500μg-1mg protein extract)

  • Compare direct antibody addition versus pre-binding to protein A/G beads

  • Consider cross-linking the antibody to beads to prevent co-elution

• Washing conditions:

  • Implement a stepwise washing strategy with decreasing detergent/salt concentrations

  • Optimize number of washes (typically 4-6) to balance removal of non-specific binding with retention of specific interactions

• Elution methods:

  • Compare different elution conditions (low pH, high pH, SDS, or peptide competition)

  • For mass spectrometry applications, consider on-bead digestion to minimize contaminants

• Controls:

  • Always include a pre-immune serum control

  • Consider using plant material where At4g02650 is minimally expressed or knockout lines

  • Perform reverse immunoprecipitation to validate interactions

How can CRISPR-based gene tagging be combined with At4g02650 antibody for functional studies?

Recent advances in CRISPR-based gene tagging offer powerful approaches for studying At4g02650 function when combined with antibody-based detection:

• Endogenous tagging strategy:

  • Use CRISPR/Cas9 to insert epitope tags (HA, FLAG, GFP) at the C-terminus of endogenous At4g02650

  • Compare protein localization and expression using both the At4g02650 antibody and tag-specific antibodies

  • This cross-validation approach enhances confidence in antibody specificity

• GAL4-UAS reporter system integration:

  • Utilize CRISPR techniques to insert T2AGAL4 or KozakGAL4 cassettes as described in the expanded toolkit for Drosophila gene tagging

  • Apply similar approaches to replace or tag At4g02650 in Arabidopsis

  • This allows visualization of gene expression patterns without relying solely on antibody detection

• Combinatorial approaches:

  • Generate knock-in reporter lines for live imaging

  • Use the At4g02650 antibody for biochemical validation

  • This multi-modal approach provides complementary data on protein dynamics

These CRISPR-based approaches can significantly enhance studies of At4g02650 function, particularly in specialized tissues like the female gametophyte where expression has been detected .

What are the considerations for applying diffusion-based protein design to improve At4g02650 antibody specificity?

Advanced protein design methodologies, such as those using diffusion probabilistic models, could potentially be applied to improve At4g02650 antibody specificity:

• Epitope optimization:

  • Use diffusion-based protein design approaches similar to those described for antigen-specific antibody design

  • Model the interaction between the At4g02650 protein and antibody binding regions

  • Identify unique epitopes that could be targeted for more specific antibody generation

• Complementarity-determining region (CDR) enhancement:

  • Apply deep generative models that jointly model sequences and structures of CDRs

  • This could allow rational design of next-generation antibodies with enhanced specificity for At4g02650

  • Such approaches would require detailed structural information about the At4g02650 protein

• Side-chain optimization:

  • Leverage equivariant neural networks to optimize side-chain orientations in antibody design

  • This atomic-resolution approach could significantly reduce cross-reactivity with similar plant proteins

While these technologies are emerging and currently more developed for therapeutic antibodies , their application to research antibodies like At4g02650 represents an exciting frontier for improving research tools in plant biology.