ORLIKE Antibody

What is ORLIKE Antibody and what is its target protein?

ORLIKE Antibody is a polyclonal antibody raised in rabbits against the Protein ORANGE-LIKE (ORLIKE), a chloroplastic protein found in Arabidopsis thaliana. The target protein is a DnaJ-like cysteine-rich domain-containing protein encoded by the ORLIKE gene located at locus At5g06130. The antibody specifically recognizes this protein in plant tissues, allowing for its detection and study in various experimental contexts. Applications for this antibody include ELISA (enzyme-linked immunosorbent assay) and Western blot techniques for protein identification and quantification .

How does the ORLIKE protein differ structurally and functionally from the related OR protein?

The ORLIKE protein (At5g06130) shares structural similarities with the OR protein (At5g61670), particularly in their DnaJ-like cysteine-rich domains, but they have distinct functions. Both are chloroplast-targeted proteins, but OR is primarily involved in carotenoid accumulation and chromoplast biogenesis, while ORLIKE's specific functions are still being characterized. The sequence homology between these proteins suggests evolutionary divergence from a common ancestor, with ORLIKE potentially representing a specialized functional adaptation within the chloroplast protein network .

What are the appropriate positive and negative controls when using ORLIKE Antibody?

For rigorous experimental design with ORLIKE Antibody, researchers should implement several controls:

Positive Controls:

• Recombinant ORLIKE protein expressed in bacterial or plant systems

• Wild-type Arabidopsis thaliana tissue extracts (particularly leaf tissue)

• Chloroplast-enriched fractions from appropriate plant tissues

Negative Controls:

• ORLIKE knockout/knockdown plant lines (CRISPR-Cas9 generated or T-DNA insertion)

• Pre-immune serum at the same concentration as the antibody

• Antibody pre-absorbed with immunizing peptide/protein

• Non-plant tissue extracts

These controls help validate signal specificity and distinguish true target recognition from background or cross-reactivity, essential for reliable data interpretation.

What is the recommended protocol for Western blot analysis using ORLIKE Antibody?

For optimal Western blot results with ORLIKE Antibody, follow this detailed protocol:

• Sample Preparation:

  • Extract total protein from plant tissues using buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitors

  • For chloroplast proteins, consider using chloroplast isolation procedures

  • Quantify protein and standardize loading (25-30 μg per lane)

• Electrophoresis and Transfer:

  • Separate proteins on 10-12% SDS-PAGE gels

  • Transfer to PVDF or nitrocellulose membrane (25V overnight at 4°C)

  • Verify transfer with Ponceau S staining

• Antibody Incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Incubate with ORLIKE Antibody at 1:1000-1:2000 dilution overnight at 4°C

  • Wash 3-4 times with TBST (10 minutes each)

  • Incubate with HRP-conjugated anti-rabbit secondary antibody (1:5000-1:10000) for 1 hour

  • Wash 5 times with TBST

• Detection and Analysis:

  • Develop using enhanced chemiluminescence (ECL) detection system

  • Expected molecular weight of ORLIKE protein is approximately 35-40 kDa

  • Include molecular weight markers and appropriate controls

  • For quantification, use image analysis software with internal loading controls

How can ORLIKE Antibody be optimized for immunolocalization studies in plant tissues?

For successful immunolocalization of ORLIKE protein in plant tissues, implement this methodological approach:

• Tissue Fixation and Processing:

  • Fix freshly harvested tissues in 4% paraformaldehyde in PBS (pH 7.4) for 2-4 hours

  • Dehydrate through an ethanol series (30%, 50%, 70%, 85%, 95%, 100%)

  • Embed in paraffin or LR White resin for thin sectioning

  • Cut sections at 5-8 μm thickness and mount on adhesive slides

• Antigen Retrieval and Blocking:

  • Perform antigen retrieval using 10 mM sodium citrate buffer (pH 6.0) at 90°C for 10-20 minutes

  • Block with 5% normal goat serum and 1% BSA in PBS with 0.1% Triton X-100 for 1 hour

  • For chloroplast proteins, consider higher detergent concentrations to improve accessibility

• Antibody Application:

  • Apply ORLIKE Antibody at 1:100-1:500 dilution in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Wash 3-4 times with PBS containing 0.1% Tween-20

  • Apply fluorophore-conjugated secondary antibody (1:200-1:500) for 1-2 hours at room temperature

  • Include DAPI (1 μg/ml) for nuclear staining

• Visualization and Confocal Microscopy:

  • Use chlorophyll autofluorescence as a chloroplast marker

  • Analyze using confocal laser scanning microscopy

  • Capture z-stack images to ensure comprehensive visualization

  • Compare signal distribution with chloroplast markers

  • Include all appropriate controls in parallel sections

What are effective strategies for troubleshooting weak or non-specific signals when using ORLIKE Antibody?

When encountering signal issues with ORLIKE Antibody, implement these systematic troubleshooting approaches:

For Weak or Absent Signal:

• Increase protein loading (30-50 μg)

• Reduce antibody dilution (try 1:500 instead of 1:2000)

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

• Use more sensitive detection systems (high-sensitivity ECL substrates)

• Optimize protein extraction methods for chloroplast proteins

• Verify protein transfer with reversible staining (Ponceau S)

For High Background or Non-specific Signals:

• Increase blocking time and concentration (try 5% BSA instead of milk)

• Add 0.1-0.3% SDS to wash buffer to reduce non-specific binding

• Increase wash duration and number (5 washes, 10 minutes each)

• Perform antibody pre-absorption with non-target proteins

• Use freshly prepared buffers and reagents

• Verify antibody specificity with peptide competition assays

For Multiple Bands:

• Use fresh samples with complete protease inhibitor cocktails

• Compare with ORLIKE knockout samples to identify specific bands

• Validate with recombinant ORLIKE protein as size reference

• Consider possible post-translational modifications or splice variants

• Optimize gel percentage and running conditions for better resolution

How can ORLIKE Antibody be utilized in protein-protein interaction studies?

For investigating ORLIKE protein interactions, employ these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Prepare non-denaturing plant lysates from appropriate tissues

  • Pre-clear lysate with Protein A/G beads to reduce non-specific binding

  • Incubate with ORLIKE Antibody (5-10 μg per mg of total protein)

  • Capture complexes with Protein A/G beads and wash extensively

  • Elute and analyze by mass spectrometry or Western blotting with antibodies against suspected interaction partners

• Proximity-dependent Biotin Identification (BioID):

  • Generate fusion constructs of ORLIKE with a promiscuous biotin ligase (BirA*)

  • Express in plant cells and supply biotin for labeling proximal proteins

  • Purify biotinylated proteins using streptavidin beads

  • Identify interacting partners by mass spectrometry

  • Validate interactions using ORLIKE Antibody in reciprocal Co-IP experiments

• Pull-down Validation:

  • Express recombinant GST- or His-tagged ORLIKE protein

  • Incubate with plant lysates or recombinant candidate interactors

  • Detect interactions by Western blot using specific antibodies

  • Perform reciprocal pull-downs to confirm binding relationships

  • Map interaction domains using truncated protein variants

What methods are available for studying post-translational modifications of ORLIKE protein using the antibody?

To investigate post-translational modifications (PTMs) of ORLIKE protein, implement these methodological approaches:

• Modification-specific Detection:

  • Immunoprecipitate ORLIKE protein using the antibody

  • Probe Western blots with antibodies against common PTMs (phosphorylation, ubiquitination, SUMOylation)

  • Use phospho-specific stains like Pro-Q Diamond for phosphorylation

  • Compare migration patterns before and after treatment with specific enzymes (phosphatases, deubiquitinases)

• Mass Spectrometry Analysis:

  • Immunoprecipitate ORLIKE using the antibody under non-denaturing conditions

  • Perform in-gel digestion with trypsin or other proteases

  • Analyze peptides by LC-MS/MS with PTM-specific enrichment methods

  • Map identified modifications to the protein sequence

  • Validate findings with targeted Western blot analysis

• In vivo Modification Dynamics:

  • Treat plants with stress conditions or signaling modulators

  • Analyze changes in ORLIKE modification patterns by Western blot

  • Compare wild-type plants with relevant kinase or PTM enzyme mutants

  • Use site-directed mutagenesis to confirm modification sites

  • Monitor modifications throughout developmental stages or stress responses

How can ORLIKE Antibody contribute to understanding chloroplast protein quality control mechanisms?

For investigating ORLIKE's role in chloroplast protein quality control, implement these research approaches:

• Stress Response Studies:

  • Subject plants to various stresses (heat, cold, high light, oxidative)

  • Monitor ORLIKE protein levels and localization using the antibody

  • Compare with known chloroplast chaperone proteins (Hsp70, ClpB3)

  • Analyze protein aggregation in wild-type versus ORLIKE mutant plants

  • Assess co-localization with protein aggregate markers under stress

• Protein Turnover Analysis:

  • Perform cycloheximide chase experiments to determine ORLIKE stability

  • Compare turnover rates under normal and stress conditions

  • Analyze ORLIKE levels in protease mutant backgrounds

  • Investigate polyubiquitination of chloroplast proteins using ORLIKE Antibody immunoprecipitation

  • Monitor degradation intermediates using the antibody

• Functional Interaction with Chaperone Networks:

  • Co-immunoprecipitate using ORLIKE Antibody to identify chaperone interactions

  • Perform split-GFP or FRET assays to confirm direct interactions

  • Compare chaperone activity in the presence/absence of ORLIKE protein

  • Assess impact on protein aggregation and disaggregation processes

  • Investigate genetic interactions between ORLIKE and chaperone/protease genes

How does ORLIKE protein function compare with other DnaJ-like proteins in chloroplast biology?

ORLIKE protein belongs to the DnaJ-like protein family but exhibits distinct characteristics compared to canonical DnaJ proteins:

Unlike canonical DnaJ proteins that function broadly as co-chaperones, ORLIKE appears to have evolved specialized functions within chloroplasts, potentially related to specific developmental processes or stress responses rather than general protein folding. This functional divergence is reflected in its unique expression patterns, interaction networks, and phenotypic effects when mutated .

What insights can ORLIKE Antibody provide regarding chloroplast development and plastid differentiation?

ORLIKE Antibody can reveal critical aspects of chloroplast development through these research approaches:

• Developmental Expression Profiling:

  • Analyze ORLIKE protein levels throughout plant development using Western blot

  • Compare expression in different tissues (cotyledons, true leaves, reproductive organs)

  • Track protein localization during proplastid-to-chloroplast differentiation

  • Correlate ORLIKE dynamics with chloroplast developmental markers

  • Investigate expression in non-green plastids (amyloplasts, chromoplasts)

• Comparative Analysis in Plastid Differentiation Mutants:

  • Examine ORLIKE expression in mutants with altered chloroplast development

  • Compare protein levels in plants grown under different light conditions

  • Analyze ORLIKE in tissues undergoing natural plastid transitions

  • Assess co-regulation with photosynthetic assembly factors

  • Investigate potential relationship with carotenoid biosynthesis pathways

• Subcellular Localization Studies:

  • Use immunogold electron microscopy with ORLIKE Antibody to determine precise sub-organellar localization

  • Perform biochemical fractionation of chloroplast sub-compartments

  • Analyze dynamic relocalization during plastid differentiation or stress

  • Compare localization patterns with related proteins like OR

  • Correlate localization with functional protein complexes

How can ORLIKE Antibody be integrated into multi-omics approaches for chloroplast research?

For comprehensive integration of ORLIKE Antibody into multi-omics research frameworks:

• Proteomics Integration:

  • Use ORLIKE Antibody for immunoprecipitation followed by mass spectrometry

  • Compare ORLIKE-associated protein complexes under different conditions

  • Correlate with global chloroplast proteome datasets

  • Identify post-translationally modified forms of ORLIKE

  • Develop quantitative Western blot protocols for validation of proteomics findings

• Transcriptomics Correlation:

  • Compare ORLIKE protein levels with transcript abundance

  • Analyze correlation between protein dynamics and gene expression changes

  • Identify discrepancies suggesting post-transcriptional regulation

  • Study impact of ORLIKE mutation on transcriptome profiles

  • Investigate potential RNA-binding capabilities of ORLIKE

• Metabolomics Connection:

  • Correlate ORLIKE protein levels with chloroplast-related metabolites

  • Compare metabolic profiles between wild-type and ORLIKE mutant plants

  • Focus on carotenoid and isoprenoid pathways potentially regulated by ORLIKE

  • Apply stable isotope labeling to track metabolic flux changes

  • Develop mathematical models integrating protein levels with metabolite dynamics

What potential applications exist for ORLIKE Antibody in studying chloroplast responses to environmental stresses?

ORLIKE Antibody offers valuable tools for investigating chloroplast stress responses:

• Abiotic Stress Response Profiling:

  • Monitor ORLIKE protein accumulation under heat, cold, drought, and oxidative stress

  • Compare protein dynamics during stress with recovery periods

  • Analyze correlation between stress severity and protein levels

  • Investigate post-translational modifications induced by specific stresses

  • Develop stress-specific expression profiles across different plant tissues

• Reactive Oxygen Species (ROS) Response Connection:

  • Study ORLIKE levels following treatment with ROS-generating compounds

  • Analyze co-localization with ROS-responsive proteins

  • Compare ORLIKE dynamics in ROS-scavenging enzyme mutants

  • Investigate potential protective functions during photooxidative stress

  • Examine relationship with chloroplast redox sensing mechanisms

• Climate Change-Related Applications:

  • Investigate ORLIKE responses under elevated CO₂ conditions

  • Analyze protein dynamics during combined heat and drought stress

  • Study potential involvement in acclimation mechanisms

  • Compare responses across ecotypes from different climatic regions

  • Develop predictive models for chloroplast protein network responses to changing environments

How might ORLIKE Antibody contribute to biotechnological applications in crop improvement?

ORLIKE Antibody research could advance agricultural biotechnology through:

• Crop Engineering Applications:

  • Use the antibody to validate ORLIKE expression in transgenic crops

  • Monitor protein levels in lines engineered for improved stress tolerance

  • Compare ORLIKE dynamics in high-yielding versus stress-resistant varieties

  • Analyze protein accumulation in crops with enhanced nutritional profiles

  • Develop screening methods for identifying promising transgenic events

• Biofortification Strategies:

  • Investigate potential connections between ORLIKE and carotenoid accumulation

  • Study relationship with vitamin biosynthesis pathways in chloroplasts

  • Monitor protein levels in biofortified crop varieties

  • Analyze impact of ORLIKE manipulation on nutritional composition

  • Develop antibody-based screening tools for biofortification programs

• Methodological Approach for Crop Applications:

  • Adapt immunological techniques for crop-specific proteins

  • Develop high-throughput screening methods using the antibody

  • Create antibody-based sensors for monitoring plant health

  • Establish cross-reactivity profiles with ORLIKE orthologs in major crops

  • Develop standardized protocols for comparative analyses across species

What considerations are important when adapting single-domain antibody technologies for ORLIKE protein research?

When applying camelid-derived single-domain antibodies (VHHs) technology to ORLIKE research:

• Advantages for Chloroplast Protein Research:

  • VHHs can recognize epitopes inaccessible to conventional antibodies

  • Their small size (15 kDa) enables better penetration into chloroplast structures

  • Greater stability under varying pH and temperature conditions

  • Potential for in vivo applications as intrabodies

  • Compatible with super-resolution microscopy techniques

• Library Development Strategy:

  • Immunize alpacas or llamas with purified recombinant ORLIKE protein

  • Perform phage display selection against native and denatured ORLIKE

  • Conduct competitive panning to identify epitope-specific binders

  • Screen for VHHs that recognize functionally relevant domains

  • Validate binding specificity against ORLIKE knockout controls

• Application-Specific Optimization:

  • Engineer VHH-Fc fusions for standard immunological applications

  • Develop fluorescently labeled VHHs for live-cell imaging

  • Create VHH-based proximity labeling tools for interactome studies

  • Optimize expression systems for different fusion constructs

  • Validate functional properties compared to conventional ORLIKE Antibody