CRRSP6 Antibody

What is CRRSP6 antibody and what systems can it be used in?

CRRSP6 antibody is a polyclonal antibody raised in rabbits against recombinant Arabidopsis thaliana CRRSP6 protein. It is specifically designed for plant research applications, with confirmed reactivity against Arabidopsis thaliana (Mouse-ear cress) samples. The antibody is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 as a preservative and 50% glycerol in 0.01M PBS at pH 7.4. It is purified using antigen affinity methods and belongs to the IgG isotype .

Applications for this antibody are primarily in research contexts requiring specific detection of CRRSP6 protein, with validated applications including ELISA and Western Blot techniques. It is important to note that this antibody is designated for research use only and should not be employed in diagnostic or therapeutic procedures .

What are the optimal storage conditions for preserving CRRSP6 antibody activity?

For optimal preservation of CRRSP6 antibody activity, storage at either -20°C or -80°C is recommended upon receipt. It is critical to avoid repeated freeze-thaw cycles as these can significantly compromise antibody functionality through protein denaturation and aggregation . For working stocks, aliquoting the antibody into smaller volumes before freezing is advisable to minimize freeze-thaw damage.

The storage buffer formulation (0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) provides stability during storage, with the glycerol component helping prevent complete freezing and the associated physical damage to antibody structure. For short-term use (1-2 weeks), refrigeration at 4°C may be suitable, but extended storage should follow the freezer temperature recommendations to maintain epitope recognition capabilities.

How should researchers validate CRRSP6 antibody specificity in their experimental systems?

Validation of CRRSP6 antibody specificity requires a multi-faceted approach:

• Positive and negative controls: Include samples from wild-type Arabidopsis thaliana (positive control) and CRRSP6 knockout lines or non-plant samples (negative controls).

• Western blot analysis: Verify that the antibody detects a band of the expected molecular weight for CRRSP6 protein. Multiple bands may indicate cross-reactivity or post-translational modifications.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide before application in your detection system. Specific binding should be significantly reduced or eliminated.

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is pulling down the target protein rather than cross-reacting with other proteins.

• Comparison with orthogonal detection methods: Correlate antibody-based detection with RNA expression data or fluorescent protein tagging where possible.

What are the recommended protocols for using CRRSP6 antibody in Western blot applications?

For optimal Western blot results with CRRSP6 antibody, the following methodological considerations are crucial:

Sample preparation:

• Extract total protein from Arabidopsis thaliana tissues using a buffer containing protease inhibitors

• Quantify protein concentration (Bradford or BCA assay) and normalize loading amounts (typically 20-50 μg per lane)

• Denature samples in Laemmli buffer (containing SDS and β-mercaptoethanol) at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Separate proteins on 10-12% SDS-PAGE gels (adjust percentage based on CRRSP6's molecular weight)

• Transfer to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight at 4°C

Antibody incubation:

• Block membrane with 5% non-fat dry milk or 3-5% BSA in TBST for 1 hour at room temperature

• Dilute primary CRRSP6 antibody 1:1000 to 1:2000 in blocking solution

• Incubate overnight at 4°C with gentle agitation

• Wash 3-4 times with TBST, 5-10 minutes each

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

• Wash 3-4 times with TBST, 5-10 minutes each

Detection:

• Apply ECL substrate and image using a digital imaging system or X-ray film

• For quantitative analysis, include loading controls (e.g., anti-actin) and use densitometry software

This protocol should be optimized for specific experimental conditions, adjusting antibody dilutions, incubation times, and washing stringency as needed to maximize signal-to-noise ratio.

How can CRRSP6 antibody be effectively used in immunolocalization studies in plant tissues?

For successful immunolocalization of CRRSP6 in plant tissues, the following protocol provides a methodological framework:

Tissue fixation and embedding:

• Fix freshly harvested tissues in 4% paraformaldehyde in PBS (pH 7.4) for 4-6 hours under vacuum

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

• Infiltrate and embed in either paraffin wax (for light microscopy) or LR White resin (for electron microscopy)

• Section tissues at 5-10 μm thickness for paraffin or 1 μm for resin

Immunostaining procedure:

• For paraffin sections: dewax in xylene and rehydrate through an ethanol series

• Perform antigen retrieval if necessary (citrate buffer pH 6.0, 95°C for 10 minutes)

• Block with 2-3% BSA, 5% normal goat serum in PBS for 1 hour

• Apply primary CRRSP6 antibody (1:100 to 1:500 dilution) and incubate overnight at 4°C in a humid chamber

• Wash extensively with PBS (3-5 times, 5-10 minutes each)

• Apply fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 488 goat anti-rabbit, 1:200-1:500) for 1-2 hours at room temperature

• Wash with PBS

• Counterstain nuclei with DAPI (1 μg/ml) for 5-10 minutes

• Mount in anti-fade medium

Controls:

• Include a negative control by omitting primary antibody

• Include tissues from CRRSP6 knockout plants if available

• Consider a peptide competition control

Confocal laser scanning microscopy is recommended for visualization, with sequential scanning to avoid bleed-through when using multiple fluorophores.

What strategies should be employed for optimizing CRRSP6 antibody dilutions in different experimental applications?

Determining optimal antibody dilutions is critical for obtaining specific signals while minimizing background. A systematic titration approach is recommended:

For Western blotting:

• Prepare a dilution series of primary antibody (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10000)

• Use identical sample loads and blotting conditions for all dilutions

• Process blots simultaneously with different antibody dilutions

• Evaluate signal-to-noise ratio for each dilution

• Select the highest dilution that produces clear specific bands with minimal background

For ELISA:

• Perform a checkerboard titration with both antigen and antibody dilutions

• Plot signal-to-noise ratios to identify optimal concentrations

• Typical starting ranges: 1:100 to 1:10,000 for primary antibody

For Immunohistochemistry/Immunofluorescence:

• Begin with manufacturer's recommended dilution range (typically 1:100 to 1:500)

• Test multiple serial dilutions on identical tissue sections

• Include appropriate controls for each dilution

• Assess specificity, signal intensity, and background staining

Optimization should be performed for each new lot of antibody, as polyclonal antibodies may exhibit batch-to-batch variation in titer and specificity.

How should researchers address non-specific binding issues when using CRRSP6 antibody?

Non-specific binding is a common challenge with antibodies that can compromise experimental interpretation. Several methodological approaches can minimize this issue:

Blocking optimization:

• Test different blocking agents (BSA, casein, normal serum, commercial blockers)

• Increase blocking time (2-3 hours or overnight at 4°C)

• Add 0.1-0.3% Triton X-100 or Tween-20 to reduce hydrophobic interactions

Antibody incubation conditions:

• Increase antibody dilution incrementally

• Add 0.1-0.5M NaCl to reduce ionic interactions

• Include 0.1% detergent in antibody dilution buffer

• Pre-absorb antibody with non-target tissues/proteins

Washing protocol enhancement:

• Increase number of washes (5-6 times)

• Extend washing duration (10-15 minutes each)

• Use more stringent washing conditions (higher salt concentration)

Additional strategies:

• For Western blots: Cut membranes to include only the region of interest

• For immunohistochemistry: Use highly purified antibody preparations

• Consider using monovalent Fab fragments if cross-linking is an issue

• Pre-clear samples before immunoprecipitation

If non-specific binding persists despite optimization, consider alternative detection methods or requesting a different lot of the antibody from the manufacturer.

What factors might cause variability in CRRSP6 detection across different experimental replicates?

Variability in CRRSP6 detection can stem from multiple sources that require systematic investigation:

Antibody-related factors:

• Batch-to-batch variation in polyclonal antibody preparations

• Degradation due to improper storage or repeated freeze-thaw cycles

• Inconsistent aliquoting leading to concentration differences

Sample preparation variables:

• Variations in protein extraction efficiency

• Incomplete protein denaturation for Western blotting

• Inconsistent fixation protocols for immunohistochemistry

• Protein modification or degradation during processing

Technical execution factors:

• Variations in incubation times and temperatures

• Inconsistent washing procedures

• Fluctuations in detection reagent activity

• Variations in equipment performance (imagers, plate readers)

Biological variables:

• Growth stage and physiological state of plant samples

• Environmental stress factors affecting protein expression

• Circadian or diurnal variations in protein levels

• Tissue-specific expression patterns

To minimize variability, implement rigorous standardization:

• Use the same antibody lot for related experiments

• Include appropriate internal controls in each experiment

• Standardize all protocols with detailed SOPs

• Process comparative samples simultaneously

• Implement appropriate normalization strategies for quantitative analyses

When reporting results, document all known sources of variation and perform sufficient biological and technical replicates to establish statistical significance.

How can researchers distinguish between genuine CRRSP6 signal and artifacts in immunofluorescence experiments?

Distinguishing genuine immunofluorescence signals from artifacts requires comprehensive controls and careful experimental design:

Essential controls:

• Omission of primary antibody: To assess non-specific binding of secondary antibody

• Pre-immune serum control: Use serum from the same animal before immunization

• Absorption control: Pre-incubate antibody with excess antigen to block specific binding

• Genetic controls: Compare wild-type to CRRSP6 knockout or knockdown tissues

• Secondary antibody only: To identify autofluorescence and non-specific binding

Technical approaches to reduce artifacts:

• Autofluorescence reduction:

  • Include tissue treatment with sodium borohydride (0.1% for 10 minutes)

  • Use Sudan Black B (0.1-0.3% in 70% ethanol) to quench plant autofluorescence

  • Implement spectral unmixing during confocal microscopy

• Cross-validation strategies:

  • Confirm localization with multiple antibodies against different epitopes

  • Use orthogonal approaches (e.g., GFP tagging, in situ hybridization)

  • Apply super-resolution microscopy techniques for detailed localization

• Image acquisition optimization:

  • Use sequential scanning in multi-channel imaging

  • Implement appropriate negative controls for threshold setting

  • Acquire z-stacks to confirm three-dimensional localization patterns

• Quantitative assessment:

  • Compare signal intensity to background in control samples

  • Apply statistical tests to determine significance of observed differences

  • Use colocalization analysis with known organelle markers

When specific signal is significantly higher than in controls and follows expected biological distribution patterns (e.g., subcellular localization consistent with protein function), confidence in genuine CRRSP6 detection is increased.

How can CRRSP6 antibody be utilized in chromatin immunoprecipitation (ChIP) experiments to study protein-DNA interactions?

While CRRSP6 antibody was primarily validated for ELISA and Western blot applications , its potential application in ChIP requires careful methodological considerations:

ChIP protocol adaptation for plant tissues:

• Tissue preparation and crosslinking:

  • Harvest 1-2g of fresh Arabidopsis tissue

  • Crosslink with 1% formaldehyde under vacuum for 10-15 minutes

  • Quench with 125mM glycine for 5 minutes

  • Wash thoroughly with ice-cold PBS

  • Flash-freeze in liquid nitrogen and grind to fine powder

• Chromatin extraction and sonication:

  • Extract chromatin in ChIP extraction buffer (50mM HEPES pH 7.5, 150mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, protease inhibitors)

  • Sonicate to achieve DNA fragments of 200-500bp (optimize sonication time and amplitude)

  • Centrifuge to remove debris and save input sample (5-10%)

• Immunoprecipitation with CRRSP6 antibody:

  • Pre-clear chromatin with protein A/G beads

  • Incubate cleared chromatin with 2-5μg CRRSP6 antibody overnight at 4°C

  • Add protein A/G beads and incubate 2-3 hours

  • Perform sequential washes with increasing stringency

  • Elute protein-DNA complexes and reverse crosslinks (65°C overnight)

  • Purify DNA using column purification

• Analysis:

  • Perform qPCR with primers targeting regions of interest

  • Calculate enrichment relative to input and IgG control

  • For genome-wide studies, prepare libraries for ChIP-seq

Validation requirements:

• Confirm antibody specificity in ChIP conditions

• Establish optimal antibody-to-chromatin ratios

• Include appropriate controls (IgG, input, positive/negative genomic regions)

• Validate enriched regions by independent methods

ChIP applications often require higher antibody concentrations than Western blotting, so optimization of antibody amount is critical for successful experiments.

What methodologies should be employed for quantitative analysis of CRRSP6 protein levels in different plant tissues?

Quantitative analysis of CRRSP6 protein levels requires a systematic approach combining proper sampling, extraction, and detection methodologies:

1. Tissue preparation and protein extraction:

• Harvest tissues at consistent developmental stages and time points

• Sample multiple biological replicates (minimum n=3)

• Extract total protein using optimized buffer (e.g., 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, protease inhibitors)

• Determine protein concentration by Bradford or BCA assay

• Normalize all samples to equal protein concentration

2. Quantitative Western blot analysis:

• Load equal amounts of total protein (20-50μg) alongside a dilution series of recombinant CRRSP6 standard

• Include housekeeping protein controls (e.g., actin, tubulin, GAPDH)

• Transfer using a system that ensures quantitative transfer (validate with Ponceau staining)

• Use fluorescent secondary antibodies for wider dynamic range and better quantification

• Image using a calibrated fluorescence scanner or digital imaging system

• Analyze using appropriate software with background subtraction

3. ELISA-based quantification:

• Develop a sandwich ELISA using CRRSP6 antibody as capture or detection antibody

• Generate a standard curve using purified recombinant CRRSP6 protein

• Ensure samples fall within the linear range of the standard curve

• Run technical triplicates for all samples and standards

• Calculate concentration using four-parameter logistic regression

4. Mass spectrometry-based approaches:

• Implement targeted proteomics (SRM/MRM) for absolute quantification

• Use isotopically labeled peptide standards corresponding to unique CRRSP6 peptides

• Extract proteins and perform tryptic digestion

• Quantify using triple quadrupole mass spectrometry

• Calculate protein abundance based on standard curves

Comparative analysis table:

For most accurate results, combine multiple methodologies and include appropriate controls and statistical analyses.

How can co-immunoprecipitation experiments be designed to study CRRSP6 protein-protein interactions in Arabidopsis thaliana?

Co-immunoprecipitation (Co-IP) experiments to study CRRSP6 protein-protein interactions require careful experimental design and extensive controls:

Experimental protocol:

• Tissue preparation and protein extraction:

  • Harvest 3-5g of Arabidopsis tissue under appropriate conditions

  • Grind in liquid nitrogen to fine powder

  • Extract in mild lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5-1% NP-40 or Triton X-100, 1mM EDTA, protease inhibitors)

  • Centrifuge at 14,000g for 15 minutes at 4°C

  • Pre-clear lysate with protein A/G beads for 1 hour at 4°C

• Immunoprecipitation:

  • Incubate pre-cleared lysate with 2-5μg CRRSP6 antibody overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate for 2-3 hours at 4°C

  • Collect beads by centrifugation

  • Wash 4-5 times with wash buffer (lysis buffer with reduced detergent)

  • Elute protein complexes with SDS sample buffer or by competitive elution

• Analysis of interacting partners:

  • Separate eluted proteins by SDS-PAGE

  • Analyze by:
    a) Western blotting with antibodies against suspected interacting partners
    b) Mass spectrometry for unbiased identification of interacting proteins

Critical controls:

• Input control: Analysis of pre-IP lysate to confirm target protein presence

• IgG control: Parallel IP with non-specific IgG from the same species

• Reverse Co-IP: Immunoprecipitate with antibodies against suspected interacting partners

• Negative control tissue: Use CRRSP6 knockout/knockdown plants

• DNase/RNase treatment: To exclude DNA/RNA-mediated interactions

Validation strategies:

• Confirm interactions through alternative methods (yeast two-hybrid, FRET, BiFC)

• Demonstrate biological relevance through functional assays

• Map interaction domains using truncation or mutation constructs

• Determine interaction specificity through competition experiments

Addressing potential artifacts:

• Use chemical crosslinking to stabilize transient interactions

• Consider native PAGE for intact complex analysis

• Test interaction dependency on specific conditions (salt concentration, pH, cofactors)

• Validate interactions in vivo using split-reporter systems

For comprehensive interactome analysis, combine Co-IP with quantitative proteomics approaches like SILAC or TMT labeling to distinguish specific from non-specific interactions with statistical confidence.

How should researchers interpret contradictory results between CRRSP6 protein levels and gene expression data?

Discrepancies between protein levels and gene expression data are common in biological systems and require careful analysis:

Potential biological explanations:

• Post-transcriptional regulation:

  • microRNA-mediated mRNA degradation

  • RNA binding protein-mediated stabilization or destabilization

  • Alternative splicing affecting protein production

  • Differences in mRNA translation efficiency

• Post-translational regulation:

  • Variations in protein half-life due to ubiquitination or other degradation signals

  • Protein stabilization through complex formation or post-translational modifications

  • Protein compartmentalization affecting antibody accessibility

  • Conditional protein degradation pathways

• Temporal dynamics:

  • Time lag between transcription and translation

  • Different turnover rates for mRNA versus protein

  • Sampling at different points in regulatory cycles

Methodological considerations:

• Technical artifacts:

  • Antibody cross-reactivity or specificity issues

  • RNA quality or primer specificity problems

  • Sample processing differences between protein and RNA workflows

  • Different sensitivities of detection methods

• Experimental design factors:

  • Cell/tissue heterogeneity in samples

  • Developmental or environmental variables

  • Circadian or other temporal effects

Resolution strategies:

• Validation experiments:

  • Perform time-course analyses to capture temporal relationships

  • Use multiple antibodies targeting different epitopes

  • Employ orthogonal protein detection methods

  • Use reporter gene constructs to monitor transcription and translation separately

• Comprehensive analysis:

  • Incorporate translatomics (ribosome profiling) to assess translation efficiency

  • Measure protein stability through cycloheximide chase experiments

  • Assess post-translational modifications with specific antibodies

  • Monitor subcellular localization changes

When interpreting contradictory results, consider that such discrepancies often reveal important biological regulatory mechanisms rather than experimental errors, and may lead to novel insights into CRRSP6 regulation.

What are the most effective experimental designs for studying CRRSP6 function in stress response pathways?

Elucidating CRRSP6 function in stress response pathways requires multi-faceted experimental approaches:

Genetic manipulation strategies:

• Loss-of-function approaches:

  • CRISPR/Cas9-mediated knockout lines

  • T-DNA insertion mutants (if available)

  • RNA interference (RNAi) for partial knockdown

  • Inducible silencing systems for temporal control

• Gain-of-function approaches:

  • Constitutive overexpression under CaMV 35S promoter

  • Inducible overexpression systems (e.g., estradiol-inducible)

  • Tissue-specific expression using appropriate promoters

  • Expression of tagged versions for localization studies

Stress exposure experimental design:

• Controlled stress application:

  • Precise control of stress intensity and duration

  • Gradual versus acute stress imposition

  • Single versus combined stresses

  • Recovery phases after stress removal

• Stress types to investigate:

  • Abiotic: drought, salinity, temperature, oxidative, light

  • Biotic: pathogens, herbivores, beneficial microbes

  • Physiological: nutrient deficiency, hormonal treatments

Comprehensive phenotyping:

• Physiological parameters:

  • Growth metrics (biomass, height, leaf area)

  • Photosynthetic parameters (quantum yield, electron transport rate)

  • Water relations (relative water content, stomatal conductance)

  • Metabolite profiles (primary/secondary metabolites)

• Cellular and molecular phenotypes:

  • Reactive oxygen species detection

  • Cell death quantification

  • Hormone level measurements

  • Protein post-translational modifications

Molecular interaction analysis:

• Temporal expression profiling:

  • Time-course analysis of gene/protein expression after stress

  • Correlation with physiological responses

  • Comparison between different tissues/cell types

• Regulatory network analysis:

  • Transcription factor binding studies (ChIP-seq)

  • Protein-protein interaction networks (Co-IP, Y2H)

  • Genetic interaction studies (double mutants, suppressors)

Statistical considerations:

• Experimental design rigor:

  • Adequate biological replication (n≥5 for most physiological assays)

  • Inclusion of appropriate controls for each genotype/treatment

  • Randomized block designs to control for environmental variation

  • Power analysis to determine sample size

• Data analysis approaches:

  • ANOVA with appropriate post-hoc tests for multiple comparisons

  • Multivariate analyses for complex phenotypic datasets

  • Correlation analyses between molecular and physiological parameters

This comprehensive experimental approach allows for robust functional characterization of CRRSP6 in stress response contexts.

What considerations are important when developing CRRSP6-targeting approaches for agricultural applications?

Translating CRRSP6 research into agricultural applications requires careful consideration of multiple factors spanning basic science to field implementation:

Mechanistic understanding prerequisites:

• Functional characterization depth:

  • Comprehensive understanding of CRRSP6's role in plant physiology

  • Identification of downstream pathways and regulatory networks

  • Characterization across multiple environmental conditions

  • Potential pleiotropic effects of manipulation

• Genetic diversity considerations:

  • Conservation of CRRSP6 function across crop species

  • Allelic variation affecting protein function or regulation

  • Potential redundancy with paralogs or related proteins

  • Species-specific regulatory mechanisms

Genetic modification strategies:

• Transgenic approaches:

  • Promoter selection for optimal expression patterns

  • Codon optimization for target crop species

  • Selection of appropriate terminators and regulatory elements

  • Consideration of position effects and transgene stability

• Gene editing considerations:

  • CRISPR/Cas9 target site selection to minimize off-targets

  • HDR versus NHEJ strategies based on desired outcome

  • Multiplex editing for pathway engineering

  • Regulatory status of gene-edited crops in target markets

Phenotypic evaluation framework:

• Controlled environment testing:

  • Standardized stress protocols mimicking field conditions

  • Multiple stress combinations and intensities

  • Assessment across developmental stages

  • Yield component analysis under stress

• Field trial design:

  • Multi-location trials across diverse environments

  • Multiple growing seasons to capture year-to-year variation

  • Appropriate statistical design (RCBD, split-plot)

  • Comprehensive agronomic trait assessment

Potential trade-offs and limitations:

• Yield-stress resistance balance:

  • Potential growth penalties under non-stress conditions

  • Impact on reproductive development and yield components

  • Effects on crop quality parameters

  • Energy allocation trade-offs

• Environmental interaction factors:

  • Genotype × environment interactions

  • Climate change adaptation considerations

  • Soil type and microbiome interactions

  • Management practice dependencies

Regulatory and commercialization pathway:

• Regulatory considerations:

  • Molecular characterization requirements

  • Food and environmental safety assessments

  • Intellectual property protection strategy

  • Regulatory differences across markets

• Technology deployment strategy:

  • Integration with existing germplasm

  • Stacking with complementary traits

  • Seed production and quality considerations

  • Farmer education and adoption strategy

Successful agricultural implementation requires interdisciplinary collaboration between molecular biologists, plant physiologists, agronomists, and regulatory specialists to navigate the complex path from laboratory findings to field application.