At5g48750 Antibody

Basic Research Questions

• What is the At5g48750 protein and why would researchers study it?

  The At5g48750 gene in Arabidopsis thaliana encodes a cytochrome b561/ferric reductase transmembrane protein with a DOMON-related domain . This protein likely functions in electron transfer processes and may play a role in iron reduction and transport within the plant. As a transmembrane protein involved in redox processes, it represents an important component of plant iron homeostasis and potential stress responses.

  Research on At5g48750 is valuable because:

  • It contributes to understanding iron metabolism in plants

  • The ferric reductase activity may be essential for iron acquisition

  • Its transmembrane nature suggests involvement in nutrient transport across cellular membranes

• What applications is the At5g48750 antibody validated for?

  The At5g48750 antibody from Cusabio (product code CSB-PA260475XA01DOA) has been validated for the following applications :

  Application	Validation Status
  ELISA	Validated
  Western Blot (WB)	Validated

  The antibody is designed specifically for detecting the Arabidopsis thaliana At5g48750 protein and should be used primarily in these validated applications for reliable results . This polyclonal antibody was generated using a recombinant Arabidopsis thaliana At5g48750 protein as the immunogen, which helps ensure specificity for the target protein .

• How should the At5g48750 antibody be properly stored and handled?

  Proper storage and handling of the At5g48750 antibody is critical for maintaining its activity and specificity. According to the product information :

  • Store the antibody at -20°C or -80°C upon receipt

  • Avoid repeated freeze-thaw cycles which can degrade antibody performance

  • The antibody is supplied in liquid form, preserved with 0.03% Proclin 300

  • The storage buffer consists of 50% glycerol and 0.01M PBS at pH 7.4

  • The antibody has been purified using antigen affinity methods

  When working with the antibody, always use clean techniques, wear gloves, maintain cold chain when possible, and aliquot the antibody upon first thaw to prevent repeated freeze-thaw cycles for the stock solution.

• What controls should be included when using the At5g48750 antibody?

  When conducting experiments with the At5g48750 antibody, the following controls should be included:

  • Positive control: Wild-type Arabidopsis thaliana tissue samples known to express At5g48750

  • Negative control: Either knockout/knockdown lines of At5g48750 or non-plant samples

  • Secondary antibody only control: Omit the primary antibody to check for non-specific binding

  • Blocking peptide control: Pre-incubate the antibody with excess recombinant At5g48750 protein

  • Loading control: Include detection of a housekeeping protein (e.g., actin) in Western blots

  These controls help validate antibody specificity and ensure experimental reliability, which is particularly important when working with plant proteins that may have homologs or related family members .

Advanced Research Questions

• How can I validate the specificity of the At5g48750 antibody for my research?

  Rigorous validation of antibody specificity is crucial for meaningful research outcomes. For the At5g48750 antibody, consider these validation approaches:

  1. Western blot analysis:

     • Compare wild-type vs. At5g48750 knockout/knockdown plants

     • Verify the presence of a single band at the expected molecular weight (check UniProt Q9FKC1)

     • Test across different tissue types to confirm expression patterns

  2. Immunoprecipitation followed by mass spectrometry:

     • Perform IP using the At5g48750 antibody

     • Analyze pulled-down proteins by MS to confirm target identity

     • Check for co-immunoprecipitating partners that align with known biology

  3. Recombinant protein competition:

     • Pre-incubate antibody with purified recombinant At5g48750 protein

     • Compare signal with and without competition

     • Signal should be significantly reduced when antibody is pre-absorbed

  4. Immunohistochemistry correlation:

     • Compare protein localization with known mRNA expression patterns

     • Use GFP-tagged At5g48750 expressed in plants as a reference

  These methods, when used in combination, provide strong evidence for antibody specificity . Document all validation steps thoroughly for publication purposes.

• What is the recommended protocol for immunolocalization of At5g48750 in plant tissues?

  For successful immunolocalization of At5g48750 in Arabidopsis tissues, follow this optimized protocol:

  Sample preparation:

  1. Fix freshly harvested tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2 hours at room temperature

  2. Wash 3x in PBS (15 minutes each)

  3. Dehydrate through an ethanol series (30%, 50%, 70%, 90%, 100%)

  4. Embed in paraffin or resin depending on the desired resolution

  5. Section at 5-10 μm thickness

  Immunolabeling:

  1. Deparaffinize and rehydrate sections

  2. Perform antigen retrieval: Citrate buffer (pH 6.0) at 95°C for 20 minutes

  3. Block with 2% BSA, 0.3% Triton X-100 in PBS for 1 hour

  4. Incubate with At5g48750 antibody (diluted 1:100-1:500) overnight at 4°C

  5. Wash 3x with PBS (10 minutes each)

  6. Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

  7. Wash 3x with PBS (10 minutes each)

  8. Counterstain nuclei with DAPI (1 μg/ml) for 10 minutes

  9. Mount with anti-fade mounting medium

  Parallel controls should include secondary-only samples and pre-absorbed primary antibody controls. Consider co-localization with organelle markers to confirm subcellular distribution, especially since At5g48750 is expected to be membrane-associated .

• How does the At5g48750 protein relate to iron transport in Arabidopsis?

  The At5g48750 protein belongs to the cytochrome b561/ferric reductase family and likely plays a significant role in iron homeostasis in Arabidopsis thaliana:

  1. Functional context: As a cytochrome b561 with ferric reductase domains, it likely participates in electron transport processes coupled to iron reduction . This is crucial because plants primarily take up iron in its reduced Fe(II) form.

  2. Physiological role: Based on related proteins in the ferric reductase family, At5g48750 may reduce Fe(III) to Fe(II) at biological membranes, facilitating iron transport across these barriers .

  3. Integration with iron transport mechanisms: Research on related proteins suggests At5g48750 may function alongside:

     • IRON-REGULATED TRANSPORTER1 (IRT1)

     • FERRIC REDUCTASE OXIDASE2 (FRO2)

     • Natural Resistance-Associated Macrophage Protein (NRAMP) transporters

  4. Regulation: The expression of At5g48750 may be responsive to:

     • Iron deficiency conditions

     • Phosphate availability, which affects iron solubility

     • Oxidative stress

  5. Subcellular localization: The protein likely functions at the plasma membrane or in intracellular membranes such as the tonoplast or endoplasmic reticulum, where it would participate in iron mobilization between cellular compartments.

  Understanding the exact role of At5g48750 requires further experimental validation using the antibody in conjunction with physiological and genetic approaches. Researchers should consider comparing wild-type and mutant plants under various iron conditions to elucidate its specific function in iron homeostasis .

• What strategies can I use to troubleshoot weak or nonspecific signals with the At5g48750 antibody?

  When experiencing issues with the At5g48750 antibody, consider these troubleshooting approaches:

  For weak signals:

  1. Antibody concentration optimization:

     • Test a dilution series (e.g., 1:100, 1:250, 1:500, 1:1000)

     • Document signal-to-noise ratio at each concentration

  2. Protein extraction improvements:

     • Use extraction buffers with various detergents (CHAPS, NP-40, Triton X-100)

     • Add protease inhibitor cocktails to prevent degradation

     • Test membrane protein enrichment protocols

  3. Enhanced detection methods:

     • Try signal amplification systems (TSA/CARD)

     • Use high-sensitivity ECL substrates for Western blots

     • Increase exposure time (with appropriate controls)

  For nonspecific signals:

  1. Blocking optimization:

     • Test different blocking agents (BSA, milk, commercial blockers)

     • Increase blocking time or concentration

  2. Washing optimization:

     • Increase washing duration or buffer stringency

     • Use PBS-T with higher Tween-20 concentration (0.1% to 0.3%)

  3. Antibody pre-absorption:

     • Pre-incubate with plant extracts from non-Arabidopsis species

     • Use knockout tissue lysates for pre-absorption if available

  4. Tissue preparation:

     • Optimize fixation conditions for immunohistochemistry

     • Test fresh vs. frozen vs. fixed samples for protein extraction

  Technical validation table:

  Issue	Possible Cause	Troubleshooting Approach
  Multiple bands	Cross-reactivity	Pre-absorb antibody; increase washing stringency
  No signal	Protein degradation	Add fresh protease inhibitors; keep samples cold
  High background	Insufficient blocking	Increase blocking time; try different blocking agents
  Variable results	Antibody degradation	Aliquot antibody; avoid freeze-thaw cycles
  Weak signal	Low protein abundance	Increase sample concentration; enrich for membrane fraction

  Document all optimization steps methodically to establish a reliable protocol for your specific experimental system .

• Can the At5g48750 antibody be used for co-immunoprecipitation studies?

  The At5g48750 antibody can potentially be used for co-immunoprecipitation (co-IP) studies to identify protein interaction partners, though this application requires careful optimization. Here's a methodological approach:

  1. Preliminary validation:

  • Confirm the antibody can recognize native (non-denatured) At5g48750 protein

  • Verify antibody affinity is sufficient for immunoprecipitation

  • Test antibody performance in IP-Western blot with the target protein

  2. Optimized co-IP protocol:

  Sample preparation:

  • Harvest fresh plant tissue and grind in liquid nitrogen

  • Extract proteins in a gentle lysis buffer (e.g., 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, protease inhibitor cocktail)

  • Clarify lysate by centrifugation (14,000 × g, 15 min, 4°C)

  Immunoprecipitation:

  • Pre-clear lysate with protein A/G beads (1 hour, 4°C)

  • Add At5g48750 antibody (2-5 μg per mg of total protein)

  • Incubate overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate 2-4 hours at 4°C

  • Wash beads 5× with washing buffer (lysis buffer with reduced detergent)

  • Elute proteins with SDS sample buffer or acid elution

  Analysis:

  • Analyze by Western blot for known/suspected interaction partners

  • For unbiased discovery, perform mass spectrometry analysis

  Controls to include:

  • Input sample (pre-IP lysate)

  • IgG control (non-specific rabbit IgG)

  • Bead-only control (no antibody)

  • When possible, immunoprecipitation from knockout/knockdown plants

  For transmembrane proteins like At5g48750, special consideration must be given to membrane solubilization conditions to maintain protein-protein interactions while efficiently extracting the protein from the membrane .

• How can I use the At5g48750 antibody to study protein regulation under different environmental stresses?

  The At5g48750 antibody can be a powerful tool for investigating protein regulation under environmental stresses, especially those affecting iron homeostasis. Here's a comprehensive approach:

  1. Experimental design for stress conditions:

  Stress Condition	Application	Duration	Control
  Iron deficiency	No Fe in nutrient solution	3, 7, 14 days	Complete nutrient solution
  Iron excess	500 μM Fe-EDTA	24, 48, 72 hours	Standard Fe (50 μM)
  Phosphate starvation	No phosphate	3, 7, 14 days	Complete nutrient solution
  Oxidative stress	H₂O₂ (5 mM)	1, 3, 6 hours	Water treatment
  Combined stresses	Fe deficiency + P starvation	7 days	Complete nutrient solution

  2. Multi-level analysis approach:

  • Protein abundance: Western blot with At5g48750 antibody to quantify total protein levels

  • Protein localization: Immunofluorescence to track subcellular redistribution

  • Post-translational modifications: 2D gel electrophoresis followed by Western blot to detect charge/size shifts

  • Protein-protein interactions: Co-IP under different stress conditions to identify stress-specific interactors

  • Protein turnover: Cycloheximide chase assay with time-course Western blot analysis

  3. Data integration and analysis:

  • Correlate protein changes with physiological parameters (e.g., chlorophyll content, Fe content)

  • Compare protein dynamics with transcriptional changes (RT-qPCR or RNA-seq data)

  • Analyze timing of changes to establish regulatory cascades

  4. Methodological considerations:

  • Run parallel samples for transcript analysis to distinguish transcriptional vs. post-transcriptional regulation

  • Include appropriate loading controls (e.g., actin, tubulin) that remain stable under your stress conditions

  • Consider tissue-specific responses by analyzing roots and shoots separately

  • Use quantitative Western blot methods (with standard curves) for accurate protein quantification

  This comprehensive approach will provide insights into how At5g48750 protein levels, localization, and interactions are modulated under stress conditions, potentially revealing its role in stress adaptation mechanisms related to iron homeostasis and membrane transport .

• How should I design experiments to study At5g48750 protein interaction with iron and electron transport chains?

  To investigate the mechanistic role of At5g48750 in iron metabolism and electron transport, the following experimental design is recommended:

  1. Protein-iron interaction studies:

  • Direct binding assays:

    • Purify recombinant At5g48750 protein (consider using only the soluble domains if full-length is challenging)

    • Perform isothermal titration calorimetry (ITC) with ferric and ferrous iron

    • Use microscale thermophoresis (MST) to determine binding constants

  • Iron reduction activity:

    • Isolate membrane fractions containing At5g48750

    • Measure ferric reduction activity using ferrozine assay

    • Compare activity in wild-type vs. At5g48750 mutant plants

  2. Electron transport analysis:

  • In vitro electron transfer:

    • Reconstitute At5g48750 in liposomes

    • Measure electron transfer using artificial electron donors/acceptors

    • Determine redox potential with cyclic voltammetry

  • In vivo electron transport chain integration:

    • Use specific inhibitors of electron transport components

    • Measure impact on At5g48750-mediated iron reduction

    • Monitor redox state changes with redox-sensitive fluorescent proteins

  3. Structural and functional analysis:

  • Domain function mapping:

    • Generate constructs with mutations in key domains:

      • DOMON domain (substrate recognition)

      • Transmembrane cytochrome b561 (electron transfer)

      • Ferric reductase domain

    • Express in At5g48750 knockout background

    • Analyze protein function with the At5g48750 antibody

  • Protein-protein interaction network:

    • Perform BioID or proximity labeling to identify proteins in close proximity to At5g48750

    • Validate key interactions with co-IP using the At5g48750 antibody

    • Map interaction domains through deletion constructs

  4. Physiological relevance:

  • Iron uptake and distribution:

    • Measure iron content in different tissues and subcellular compartments

    • Use Perls' staining and ferrozine assays to quantify iron forms

    • Compare wild-type and At5g48750 mutant plants under varying iron conditions

  • Integration with known iron homeostasis pathways:

    • Analyze genetic interactions with known iron transport genes (IRT1, FRO2)

    • Create double mutants and analyze phenotypes

    • Use the At5g48750 antibody to check protein expression in different genetic backgrounds

  This comprehensive approach combines biochemical, structural, and physiological methods to elucidate how At5g48750 participates in iron metabolism and electron transport, providing mechanistic insights into its function .

• What techniques can be used to quantify At5g48750 protein expression levels across different tissues and developmental stages?

  To comprehensively quantify At5g48750 protein expression across tissues and developmental stages, employ these methodological approaches:

  1. Quantitative Western blot analysis:

  • Sample preparation protocol:

    1. Collect tissues at defined developmental stages (seedling, vegetative, flowering, senescence)

    2. Separate into tissue types (roots, stems, young leaves, mature leaves, flowers, siliques)

    3. Extract proteins using a buffer optimized for membrane proteins (e.g., containing 1% SDS or 0.5% Triton X-100)

    4. Quantify total protein using BCA or Bradford assay

  • Quantitative Western blot procedure:

    1. Include recombinant At5g48750 protein standards for calibration curve (5-100 ng range)

    2. Load equal total protein amounts (30-50 μg) per lane

    3. Include a housekeeping protein control (e.g., actin) for normalization

    4. Use fluorescent secondary antibodies for wider dynamic range

    5. Analyze band intensity with image analysis software (ImageJ/FIJI)

  2. Tissue-specific expression analysis:

  • Immunohistochemistry approach:

    1. Prepare tissue sections from different organs at key developmental stages

    2. Perform immunolabeling with At5g48750 antibody

    3. Use fluorescence intensity quantification for semi-quantitative analysis

    4. Include negative controls (knockout tissue, secondary-only controls)

  • ELISA-based quantification:

    1. Develop a sandwich ELISA using At5g48750 antibody and a tagged secondary detection antibody

    2. Generate tissue-specific protein extracts

    3. Create standard curves with recombinant protein

    4. Calculate absolute protein concentrations in each sample

  3. Data normalization and analysis:

  Normalization Method	Application	Advantages	Limitations
  Total protein	Bradford/BCA assay	Simple, widely used	May vary with tissue type
  Housekeeping proteins	Actin, tubulin	Common standard	Expression may not be constant
  Protein/fresh weight	Weight normalization	Accounts for tissue density	Affected by water content
  Protein/cell number	Flow cytometry	Precise per-cell measure	Difficult for solid tissues

  4. Data integration and visualization:

  • Create heat maps of protein expression across tissues and developmental stages

  • Perform cluster analysis to identify co-regulated proteins

  • Compare with publicly available transcriptomic data to identify post-transcriptional regulation

  • Present data as fold-change relative to a reference tissue/stage

  5. Advanced approaches:

  • Single-cell analysis:

    • Use fluorescence-activated cell sorting (FACS) with protoplasts

    • Perform immunolabeling followed by flow cytometry

  • Mass spectrometry-based quantification:

    • Use targeted proteomics (PRM/MRM) for absolute quantification

    • Employ stable isotope-labeled peptide standards

  This multi-method approach provides robust quantification of At5g48750 protein across different tissues and developmental contexts, revealing spatial and temporal regulation patterns .