CRRSP35 Antibody

What is CRRSP35 and what cellular functions does it serve in Arabidopsis thaliana?

CRRSP35 is a protein found in Arabidopsis thaliana (Mouse-ear cress), a model plant organism widely used in molecular biology research. The protein belongs to a family involved in RNA processing mechanisms, particularly related to SR proteins that govern splice site selection and spliceosome assembly. SR proteins are critical for post-transcriptional regulation through alternative splicing, which is an important mechanism for fine-tuning gene expression . In Arabidopsis, there are 19 SR proteins, nearly twice as many as found in humans, which fall into seven distinct subfamilies . The specific function of CRRSP35 relates to RNA recognition and potentially to the regulation of particular alternative splicing events that are evolutionarily conserved from green algae to flowering plants.

What are the optimal storage conditions for CRRSP35 antibody to maintain its activity?

For maximum antibody stability and activity preservation, CRRSP35 antibody should be stored at either -20°C or -80°C immediately upon receipt . Repeated freeze-thaw cycles should be strictly avoided as they can denature the antibody and reduce its binding efficiency . The antibody is supplied in liquid form with a storage buffer containing 0.03% Proclin 300 (as a preservative), 50% Glycerol, and 0.01M PBS at pH 7.4 . For working solutions, store at 4°C for short-term use (up to one week). When removing aliquots for experiments, allow the antibody to warm gradually to room temperature before opening to prevent condensation, which can introduce contamination and accelerate degradation.

What validation methods confirm the specificity of CRRSP35 antibody for experimental use?

The specificity of CRRSP35 antibody is validated through multiple complementary approaches to ensure reliable experimental results:

• Immunogen Verification: The antibody is generated against a recombinant Arabidopsis thaliana CRRSP35 protein, ensuring target-specific immune response .

• Application-Specific Testing: The antibody has been validated for specific applications including ELISA and Western Blot (WB), with verification of proper antigen identification in each assay format .

• Purification Method: Antigen affinity purification ensures that only antibodies binding specifically to the target antigen are retained in the final product .

• Species Reactivity Assessment: The antibody has been specifically tested for reactivity with Arabidopsis thaliana proteins, confirming its utility in plant research applications .

For additional validation, researchers should consider performing:

• Peptide competition assays

• Knockout/knockdown cell line testing

• Cross-reactivity testing against related protein family members

What are the recommended protocols for using CRRSP35 antibody in Western Blot applications?

For optimal Western Blot results with CRRSP35 antibody, follow this detailed protocol:

• Sample Preparation:

  • Extract total protein from Arabidopsis tissue using a buffer containing 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor cocktail.

  • Determine protein concentration using Bradford or BCA assay.

  • Mix 20-40μg protein with Laemmli buffer and denature at 95°C for 5 minutes.

• Gel Electrophoresis and Transfer:

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

  • Transfer to PVDF membrane (0.45μm) at 100V for 60-90 minutes in cold transfer buffer.

• Antibody Incubation:

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

  • Dilute CRRSP35 antibody 1:1000 to 1:2000 in blocking solution.

  • Incubate membrane with primary antibody overnight at 4°C with gentle agitation.

  • Wash 3 times with TBST, 5 minutes each.

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

  • Wash 3 times with TBST, 5 minutes each.

• Detection:

  • Apply ECL substrate and capture chemiluminescent signal.

  • Expected molecular weight for CRRSP35 should be confirmed against the UniProt database (Q9LRK2) .

• Controls:

  • Include positive control (known CRRSP35-expressing tissue)

  • Include negative control (tissue with low or no CRRSP35 expression)

  • Consider loading control (anti-actin or anti-tubulin) for normalization

How can CRRSP35 antibody be optimized for immunoprecipitation of plant protein complexes?

Optimizing CRRSP35 antibody for immunoprecipitation in plant systems requires special considerations:

• Extraction Buffer Optimization:

  • Use a gentle lysis buffer: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with plant-specific protease inhibitor cocktail.

  • Include 1-2% polyvinylpyrrolidone (PVP) to remove phenolic compounds common in plant tissues.

  • Add 1mM DTT to protect from oxidation.

• Pre-clearing Step:

  • Pre-clear lysate with protein A/G beads for 1 hour at 4°C to reduce non-specific binding.

  • Centrifuge at 12,000g for 10 minutes and collect supernatant.

• Antibody Binding:

  • Use 2-5μg of CRRSP35 antibody per 500μg of total protein.

  • Incubate with rotation overnight at 4°C.

  • Add pre-washed protein A/G beads and incubate for additional 2-3 hours.

• Washing Conditions:

  • Perform 4-5 washes with increasingly stringent buffers:

    • First wash: IP buffer

    • Second wash: IP buffer with 300mM NaCl

    • Third wash: IP buffer with 0.1% SDS

    • Final wash: 50mM Tris-HCl (pH 7.5)

• Elution and Analysis:

  • Elute with 2X SDS sample buffer at 95°C for 5 minutes or with a more gentle elution using peptide competition.

  • Analyze by Western blot or mass spectrometry to identify interacting partners.

For RNA-binding proteins like CRRSP35, consider performing RNA immunoprecipitation (RIP) by adding RNase inhibitors to all buffers and processing samples for RNA isolation after protein elution.

How can CRRSP35 antibody be utilized to investigate alternative splicing mechanisms in Arabidopsis?

CRRSP35 antibody can be strategically employed to explore alternative splicing mechanisms in Arabidopsis through multiple sophisticated approaches:

• RNA Immunoprecipitation (RIP) Analysis:

  • Use CRRSP35 antibody to immunoprecipitate protein-RNA complexes in vivo.

  • Extract and analyze bound RNAs using RT-PCR with primers specific to alternatively spliced regions.

  • Sequence the recovered RNA to identify binding motifs and targets.

• Chromatin Immunoprecipitation (ChIP) Adaptations:

  • Perform ChIP-like experiments to detect co-transcriptional splicing events where CRRSP35 might bind nascent RNA.

  • Cross-link protein-RNA complexes using UV irradiation (CLIP methodology).

  • Map binding sites relative to alternatively spliced exons and introns.

• Immunofluorescence Colocalization:

  • Perform dual immunostaining with CRRSP35 antibody and other splicing factors.

  • Analyze colocalization patterns during different developmental stages or stress conditions.

  • Quantify changes in nuclear speckle formation, which are hubs of splicing activity.

• Splicing Reporter Assays:

  • Develop minigene constructs containing alternatively spliced introns similar to those found in SR protein genes .

  • Manipulate CRRSP35 levels through overexpression or silencing approaches.

  • Use the antibody to confirm expression changes and correlate with observed splicing pattern alterations.

The Arabidopsis genome encodes 19 SR proteins that fall into seven subfamilies, with three plant-specific subfamilies characterized by relatively long alternatively spliced introns in their first RNA recognition motif . By targeting CRRSP35 and analyzing changes in splicing patterns of these introns, researchers can gain insights into the regulatory networks controlling alternative splicing in plants.

What experimental approaches can determine if CRRSP35 exhibits autoregulation of alternative splicing similar to atRSZ33?

To investigate whether CRRSP35 exhibits autoregulation of alternative splicing similar to atRSZ33 (a member of the RS2Z subfamily), implement the following experimental approaches:

• Minigene Construct Analysis:

  • Create a minigene construct containing the CRRSP35 genomic region with its potentially alternatively spliced intron.

  • Co-express this construct with increasing amounts of CRRSP35 protein in Arabidopsis protoplasts.

  • Analyze splicing patterns using RT-PCR with specific primers flanking the intron of interest.

  • Compare results with known autoregulated SR proteins like atRSZ33 and non-autoregulated ones like atRSp31 .

• In Vitro Splicing Assays:

  • Generate in vitro transcripts containing the CRRSP35 alternative intron.

  • Perform in vitro splicing assays using plant nuclear extracts supplemented with recombinant CRRSP35 protein.

  • Analyze splicing products using methods similar to those described for atRSp31 .

  • Look for concentration-dependent effects on splicing patterns.

• CRISPR-Based Transcriptional Induction:

  • Develop an inducible CRISPR activation system targeting the CRRSP35 promoter.

  • Monitor changes in endogenous CRRSP35 splicing patterns following induction using RT-PCR.

  • Quantify isoform ratios using real-time PCR or RNA-Seq.

• Cross-Regulation Testing:

  • Overexpress related SR proteins (like atRSZ33) and assess their impact on CRRSP35 splicing.

  • Perform reciprocal experiments overexpressing CRRSP35 to test its effects on other SR proteins.

  • Use the antibody to confirm protein expression in all experimental conditions.

This comparative approach will help position CRRSP35 within the regulatory network of SR proteins in Arabidopsis.

What strategies can overcome high background or non-specific binding when using CRRSP35 antibody in plant tissue immunostaining?

High background and non-specific binding are common challenges when working with plant tissues due to their complex composition. To overcome these issues when using CRRSP35 antibody for immunostaining:

• Tissue Fixation and Permeabilization Optimization:

  • Test different fixatives (4% paraformaldehyde vs. methanol-acetone) to determine optimal epitope preservation.

  • Optimize permeabilization conditions using lower concentrations of detergents (0.1-0.3% Triton X-100) to reduce damage to plant cell structures.

  • Consider enzyme-mediated cell wall digestion (1-2% cellulase, 0.5% macerozyme) for improved antibody penetration.

• Blocking Enhancements:

  • Use plant-specific blocking agents: 5% normal serum (from the species of secondary antibody origin) plus 1% BSA.

  • Add 0.1-0.3% plant-derived proteins (like non-fat milk) to reduce plant-specific background.

  • Include 0.1-0.2% Tween-20 in all wash and incubation buffers.

  • Pre-absorb antibody with acetone powder prepared from non-target plant tissues.

• Antibody Dilution Optimization:

  • Perform titration experiments starting from 1:100 to 1:1000 dilutions.

  • Increase antibody incubation temperature from 4°C to 25°C while reducing incubation time.

  • Add 5-10% glycerol to antibody dilution buffer to enhance stability and specificity.

• Signal Amplification with Controls:

  • Implement tyramide signal amplification if signal is weak after background reduction.

  • Always run parallel negative controls:

    • Omission of primary antibody

    • Pre-immune serum at same concentration

    • Competitive blocking with immunizing peptide

• Data Validation:

  • Compare immunostaining patterns with in situ hybridization for CRRSP35 mRNA.

  • Use tissues from knockdown plants as specificity controls.

  • Quantify signal-to-noise ratio using digital image analysis.

How can researchers address potential cross-reactivity issues when studying CRRSP35 in the context of other SR proteins?

Addressing cross-reactivity is crucial when studying CRRSP35 within the context of the 19 SR proteins in Arabidopsis. Implement these strategies to ensure specificity:

• Epitope Analysis and Antibody Selection:

  • Perform in silico analysis comparing CRRSP35 epitopes against other SR protein sequences.

  • Select antibodies raised against unique regions rather than conserved domains like the RNA recognition motif (RRM).

  • Consider using antibodies against C-terminal regions which often show greater sequence divergence.

• Validation Through Multiple Methods:

  • Confirm specificity through Western blot analysis of recombinant SR proteins expressed in bacteria.

  • Test antibody reactivity in SR protein knockout/knockdown lines.

  • Perform immunoprecipitation followed by mass spectrometry to identify all proteins captured by the antibody.

• Blocking Strategies for Specific Applications:

  • Pre-incubate antibody with recombinant proteins of closely related SR family members.

  • Use peptide competition assays with synthetic peptides from homologous regions of related SR proteins.

  • Design a sequential immunoprecipitation strategy to deplete cross-reactive proteins.

• Alternative Detection Approaches:

  • Generate epitope-tagged CRRSP35 constructs for expression studies.

  • Develop isoform-specific RT-PCR assays to correlate protein levels with transcript expression.

  • Consider proteomics approaches with multiple reaction monitoring (MRM) for absolute quantification.

• Comprehensive Bioinformatic Analysis:

  • Conduct phylogenetic analysis of all SR proteins to identify the most closely related family members.

  • Generate sequence alignment heat maps highlighting regions of highest similarity.

  • Design experiments that can differentiate between closely related SR proteins based on known functional differences.

How evolutionarily conserved is CRRSP35 across plant species, and what implications does this have for cross-species reactivity of the antibody?

The evolutionary conservation of CRRSP35 across plant species has significant implications for antibody cross-reactivity and comparative research:

• Conservation Analysis:

  • SR proteins show remarkable conservation from green algae to flowering plants, particularly in their functional domains and alternative splicing patterns .

  • The positions of long introns capable of alternative splicing are preserved across diverse plant taxa .

  • In certain alternative splicing events, splicing signals are embedded in highly conserved sequences that occur in at least one gene per subfamily across different plant species .

• Cross-Reactivity Prediction:

  • Based on evolutionary conservation patterns, CRRSP35 antibody raised against Arabidopsis thaliana may show cross-reactivity with orthologous proteins in closely related Brassicaceae family members.

  • Decreasing reactivity would be expected along phylogenetic distance, with potentially detectable signals in other eudicots but limited reactivity in monocots or non-vascular plants.

  • The RNA recognition motif (RRM) domains are typically more conserved than the RS (arginine/serine-rich) domains, resulting in domain-specific cross-reactivity patterns.

• Experimental Validation Methods:

  • Perform Western blot analysis on protein extracts from multiple plant species to create a cross-reactivity profile.

  • Use bioinformatics to identify species with high sequence conservation at the epitope region.

  • Test immunoprecipitation efficiency across diverse plant taxa with varying evolutionary distances from Arabidopsis.

• Implications for Comparative Research:

  • Highly conserved epitopes suggest fundamental functional importance across plant evolution.

  • Conservation of alternative splicing patterns indicates preserved regulatory mechanisms.

  • Species-specific variations in reactivity may highlight functionally important adaptations in RNA processing mechanisms.

The evolutionary preservation of alternative splice forms together with highly conserved intron features argues for additional functions in plant-specific SR proteins that have been maintained throughout plant evolution . This conservation suggests that CRRSP35 likely plays a fundamental role in RNA processing that predates the divergence of major plant lineages.

What methodological approaches can determine whether CRRSP35 functions in alternative splicing regulation similar to other plant-specific SR proteins?

To determine if CRRSP35 functions in alternative splicing regulation similarly to other plant-specific SR proteins, implement these methodological approaches:

• Functional Complementation Studies:

  • Generate loss-of-function CRRSP35 mutants using CRISPR/Cas9.

  • Introduce wild-type or mutated CRRSP35 variants to test rescue capabilities.

  • Analyze global splicing patterns using RNA-Seq before and after complementation.

  • Compare results with known SR protein mutants to identify shared vs. unique splicing targets.

• Domain Swap Experiments:

  • Create chimeric proteins by swapping domains between CRRSP35 and other SR proteins like atRSZ33 or atRSp31 .

  • Express these constructs in protoplasts and analyze their effects on splicing of known target transcripts.

  • Use immunoprecipitation with CRRSP35 antibody to confirm expression and localization.

• In Vitro RNA Binding Studies:

  • Express and purify recombinant CRRSP35 protein domains.

  • Perform RNA electrophoretic mobility shift assays (EMSA) with synthetic RNA oligonucleotides.

  • Compare binding patterns with those of other SR proteins to identify shared motif preferences.

  • Use RNA compete assays to determine global RNA binding preferences.

• Splicing Reporter Systems:

  • Develop minigene constructs containing alternatively spliced introns from various target genes.

  • Co-express with CRRSP35 in plant protoplasts using methods similar to those described for atRSp31 .

  • Analyze splicing patterns through RT-PCR as described in the literature for similar SR proteins .

  • Determine dose-response relationships between CRRSP35 expression levels and target splicing outcomes.

• Interactome Analysis:

  • Perform co-immunoprecipitation using CRRSP35 antibody followed by mass spectrometry.

  • Identify interactions with other splicing factors and compare with interactome data from other SR proteins.

  • Confirm key interactions through bimolecular fluorescence complementation (BiFC) or Förster resonance energy transfer (FRET).

These approaches will collectively determine whether CRRSP35 shares functional characteristics with other plant-specific SR proteins in regulating alternative splicing and elucidate any unique properties that distinguish it within this protein family.

What emerging technologies might enhance the application of CRRSP35 antibody in plant molecular research?

Several cutting-edge technologies hold promise for advancing CRRSP35 antibody applications in plant molecular research:

• Proximity Labeling Proteomics:

  • Develop CRRSP35 fusion proteins with BioID or TurboID for in vivo proximity labeling.

  • Use antibodies to validate expression and proper localization of fusion constructs.

  • Map dynamic protein interaction networks under various developmental stages or stress conditions.

  • Identify novel components of plant-specific splicing complexes that associate with CRRSP35.

• Single-Cell Technologies:

  • Apply CRRSP35 antibody in single-cell proteomics workflows to analyze cell-type-specific expression patterns.

  • Combine with single-cell transcriptomics to correlate protein levels with splicing outcomes at cellular resolution.

  • Develop protocols for plant nuclei isolation compatible with CUT&Tag or CUT&RUN methodologies for mapping RNA-protein interactions in specific cell types.

• CRISPR-Based Epitope Tagging:

  • Use CRISPR/Cas9 to insert small epitope tags into the endogenous CRRSP35 locus.

  • Apply both CRRSP35 antibody and epitope tag antibodies for validation and cross-confirmation.

  • Enable live-cell imaging of native CRRSP35 dynamics by inserting fluorescent protein tags.

• Spatial Transcriptomics Integration:

  • Combine immunohistochemistry using CRRSP35 antibody with spatial transcriptomics.

  • Correlate protein localization with alternative splicing patterns across different tissue regions.

  • Develop computational tools to integrate protein expression data with spatially resolved RNA isoform maps.

• Nanobody Development:

  • Generate plant-optimized nanobodies against CRRSP35 for improved tissue penetration.

  • Create intrabodies for targeted manipulation of CRRSP35 function in specific subcellular compartments.

  • Develop bi-specific antibodies to study co-localization with other splicing factors.

These emerging technologies will enable more precise spatial and temporal understanding of CRRSP35's role in RNA processing and alternative splicing regulation in plants, potentially revealing new functions beyond those currently known for SR proteins.

How might comparative studies using CRRSP35 antibody contribute to understanding divergent RNA processing mechanisms between plants and other eukaryotes?

Comparative studies using CRRSP35 antibody can provide unique insights into the evolutionary divergence of RNA processing mechanisms between plants and other eukaryotes:

• Evolutionary Proteomics Approaches:

  • Apply CRRSP35 antibody in immunoprecipitation studies across diverse plant lineages where cross-reactivity permits.

  • Compare immunoprecipitated complexes through mass spectrometry to identify conserved and lineage-specific interacting partners.

  • Reconstruct the evolutionary history of plant-specific splicing complexes relative to animal and fungal systems.

• Functional Conservation Analysis:

  • Express CRRSP35 in heterologous systems (yeast, mammalian cells) to assess cross-kingdom functionality.

  • Test whether CRRSP35 can complement SR protein mutants in non-plant systems.

  • Use the antibody to confirm expression and proper localization in these heterologous systems.

  • Compare with similar experiments using human SR proteins expressed in plant systems.

• Comparative Target Recognition Studies:

  • Identify RNA targets of CRRSP35 through CLIP-seq or RIP-seq in Arabidopsis.

  • Compare binding motifs with those recognized by SR proteins in humans and other model organisms.

  • Evaluate whether plant-specific RNA structural elements influence CRRSP35 binding.

• Regulatory Network Comparisons:

  • Map the regulatory relationships between CRRSP35 and other splicing factors in plants.

  • Compare with known regulatory networks in other eukaryotes to identify plant-specific regulatory circuits.

  • Use antibody-based approaches to validate key regulatory interactions.

• Stress Response Regulatory Mechanisms:

  • Analyze CRRSP35 expression and localization during various stress conditions.

  • Compare with stress-induced changes in SR protein function in non-plant systems.

  • Test whether plant-specific stress responses involve unique RNA processing mechanisms mediated by CRRSP35 and related proteins.