CAPRIN2 Antibody, HRP conjugated

What is CAPRIN2 and what cellular functions does it regulate?

CAPRIN2 (Cytoplasmic activation/proliferation-associated protein 2, also known as RNA granule protein 140 or RNG140) is a multifunctional RNA binding protein primarily involved in:

• Regulation of arginine vasopressin (AVP) mRNA metabolism in the hypothalamus

• Modulation of fluid homeostasis through osmoregulatory mechanisms

• Enhancement of canonical Wnt signaling via LRP5/6 phosphorylation

• Development of ocular structures including the lens

Studies have demonstrated that CAPRIN2 binds to AVP mRNA in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus, regulating both its abundance and poly(A) tail length during osmotic stress . Additionally, CAPRIN2 facilitates LRP5/6 phosphorylation by glycogen synthase kinase 3 (GSK3), enhancing the interaction between Axin and LRP5/6, which is crucial for canonical Wnt signaling .

What applications are CAPRIN2 antibodies suitable for?

Based on validated research applications, CAPRIN2 antibodies have been successfully used in:

For HRP-conjugated CAPRIN2 antibodies specifically, ELISA is a primary validated application . The conjugation to HRP eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal .

What is the expected molecular weight for CAPRIN2 detection?

The calculated molecular weight of CAPRIN2 is approximately 126 kDa (1127 amino acids), but the observed molecular weight typically ranges between 126-150 kDa in SDS-PAGE analysis . This variability may reflect:

• Post-translational modifications

• Splice variants

• Tissue-specific processing

When performing western blot analysis, it's advisable to use positive control samples such as Y79 cells, SH-SY5Y cells, or HEK-293 cells, which have been confirmed to express detectable levels of CAPRIN2 .

How should I optimize immunofluorescence protocols for CAPRIN2 detection in the hypothalamus?

For optimal detection of CAPRIN2 in hypothalamic tissues (particularly in the SON and PVN regions), the following methodology has proven effective:

• Fixation and sectioning:

  • Transcardial perfusion with 4% paraformaldehyde

  • Post-fixation overnight

  • Cryoprotection in 30% sucrose for 3 days

  • Sectioning at 30 μm thickness

• Antigen retrieval:

  • Immersion in 0.1M sodium citrate (pH 6.0) at 100°C for 15 minutes

• Blocking and permeabilization:

  • 10% donkey serum in PBS containing 0.3% Triton X-100 for 30 minutes

• Antibody incubation:

  • Primary antibody in 1% normal donkey serum + 0.3% Triton X-100

  • Incubate for 1 hour at room temperature followed by overnight at 4°C

• Visualization:

  • Use appropriate fluorophore-conjugated secondary antibodies

  • Co-staining with AVP-neurophysin (NP-II) can help identify AVP-expressing neurons

This protocol has successfully demonstrated CAPRIN2 expression in AVP-producing magnocellular neurosecretory cells (MCNs) in both control and osmotically stimulated rats .

How can I design experiments to investigate CAPRIN2's role in osmotic stress response?

To study CAPRIN2's function in osmoregulation, a comprehensive experimental approach should include:

• In vivo osmotic challenge models:

  • Salt-loading (2% NaCl in drinking water for 7 days)

  • Dehydration (complete fluid deprivation for 72 hours)

  • Monitor physiological parameters: urine output, fluid intake, urine osmolality, plasma AVP levels

• Gene knockdown strategy:

  • Design shRNA targeting all known Caprin-2 transcript variants

  • Package into lentiviral vectors (including reporter genes like eGFP)

  • Stereotaxic delivery to hypothalamic nuclei (SON and PVN)

  • Use scrambled shRNA as control

• Validation of knockdown efficiency:

  • Quantitative RT-PCR for mRNA levels

  • Immunofluorescence for protein reduction

  • Compare transduced (eGFP-positive) versus non-transduced neurons

• Functional assessments:

  • Measure AVP mRNA levels and poly(A) tail length

  • Quantify plasma AVP peptide concentrations

  • Analyze physiological parameters in metabolic cages

This approach has revealed that Caprin-2 knockdown decreases urine output and fluid intake while increasing urine osmolality, urine sodium concentration, and plasma AVP levels, indicating its critical role in fluid homeostasis regulation .

What are the structural considerations when targeting specific domains of CAPRIN2 with antibodies?

CAPRIN2 contains several functional domains that should be considered when selecting antibodies:

• Homologous Region 1 (HR1) domain:

  • Located at the N-terminus

  • Forms homo-dimers resembling a pair of scissors

  • Contains a basic cluster (including R200 and R201) critical for:

    • Plasma membrane localization

    • PI4P binding

    • Wnt signaling activity

• RNA-binding regions:

  • The HR1 domain has RNA-binding capability

  • Specific for certain mRNAs like AVP mRNA

• Coiled-coil domains:

  • Important for protein-protein interactions

  • May mediate dimerization

Antibodies targeting different epitopes may yield varying results depending on:

• Domain accessibility in different experimental conditions

• Conformational changes during protein-protein interactions

• Post-translational modifications affecting epitope recognition

Researchers should select antibodies that target regions appropriate for their specific research questions (e.g., antibodies against the HR1 domain for Wnt signaling studies or RNA-binding region for AVP regulation studies) .

How can I perform RNA immunoprecipitation (RIP) assays to study CAPRIN2-mRNA interactions?

For investigating CAPRIN2-bound mRNAs, the following RIP protocol has proven effective:

• Tissue preparation:

  • Harvest tissues of interest (e.g., hypothalamic SON and PVN)

  • Homogenize in appropriate lysis buffer containing RNase inhibitors

• Immunoprecipitation:

  • Pre-clear lysates with protein A/G beads

  • Incubate with anti-CAPRIN2 antibody (typically 2-5 μg)

  • Include non-specific IgG as negative control

  • Capture antibody-protein-RNA complexes with protein A/G beads

• RNA extraction and analysis:

  • Extract RNA from immunoprecipitates

  • Perform quantitative RT-PCR for target mRNAs (e.g., AVP)

  • Calculate enrichment compared to IgG control

• Validation:

  • Include known CAPRIN2-binding mRNAs (AVP) as positive controls

  • Include non-target mRNAs (e.g., Rpl19) as negative controls

This approach has demonstrated that CAPRIN2 binds AVP mRNA at levels 1-2 orders of magnitude higher than non-specific IgG controls in both euhydrated and salt-loaded rats, confirming specific interaction .

What experimental approaches can determine if CAPRIN2 affects mRNA stability versus translation?

To distinguish between CAPRIN2's effects on mRNA stability versus translation:

• mRNA stability assessment:

  • Actinomycin D chase experiments:

    • Treat cells with actinomycin D to inhibit transcription

    • Compare mRNA decay rates in CAPRIN2 knockdown versus control cells

    • Quantify target mRNA at multiple time points by qRT-PCR

  • Poly(A) tail length analysis:

    • Northern blot analysis with and without oligo(dT) hybridization and RNase H digestion

    • Compare changes in mRNA size before and after poly(A) tail removal

    • CAPRIN2 knockdown has been shown to shorten AVP mRNA poly(A) tails

• Translation efficiency analysis:

  • Polysome profiling:

    • Fractionate cytoplasmic extracts on sucrose gradients

    • Analyze distribution of target mRNAs across non-translating, light and heavy polysome fractions

    • Compare profiles between CAPRIN2 knockdown and control conditions

  • Metabolic labeling:

    • Pulse-label cells with [35S]-methionine/cysteine

    • Immunoprecipitate protein of interest

    • Compare newly synthesized protein levels

• Recapitulated in vitro system:

  • Co-express target gene (e.g., AVP) with CAPRIN2 under heterologous promoters

  • Analyze effects on mRNA abundance and poly(A) tail length

  • Include CAPRIN2 knockdown controls

In the case of AVP, research has shown that CAPRIN2 overexpression increases AVP mRNA abundance and poly(A) tail length, while knockdown produces opposite effects, suggesting primary regulation at the mRNA stability level .

How can I distinguish between CAPRIN2 and the related protein CAPRIN1 in experimental systems?

Distinguishing between CAPRIN2 and CAPRIN1 is crucial due to their structural similarities but distinct functions:

• Antibody selection:

  • Choose antibodies raised against unique regions not conserved between CAPRIN1 and CAPRIN2

  • Verify specificity by testing in systems with selective knockdown of each protein

  • Western blot analysis should show distinct molecular weight patterns

• Functional discrimination:

  • Co-immunoprecipitation experiments show that CAPRIN2, but not CAPRIN1, interacts with LRP5

  • CAPRIN2 selectively enhances canonical Wnt signaling

  • CAPRIN2 has specific binding to AVP mRNA

• Expression pattern analysis:

  • CAPRIN2 shows tissue-specific expression patterns (e.g., enriched in lens fiber cells and hypothalamic nuclei)

  • Perform qRT-PCR using primers specific to unique regions of each transcript

  • Use immunofluorescence with validated antibodies to confirm distinct localization patterns

When conducting knockdown or overexpression studies, carefully design constructs targeting unique sequences to avoid cross-reactivity between these related proteins.

What controls should I include when studying CAPRIN2's role in Wnt signaling pathways?

For rigorous investigation of CAPRIN2's function in Wnt signaling, include the following controls:

• Pathway activation controls:

  • Positive control: Wnt-3a stimulation

  • Negative control: Wnt pathway inhibitors (e.g., DKK1)

  • Dose-response evaluation of Wnt ligands

• Protein interaction validation:

  • Co-immunoprecipitation of CAPRIN2 with LRP5/6

  • Verification that Wnt stimulation doesn't significantly affect CAPRIN2-LRP6 binding

  • In vitro binding assays with recombinant proteins to confirm direct interaction

• Functional domain analysis:

  • Mutation of key residues in the HR1 domain (R200E/R201E)

  • Assessment of membrane localization

  • Evaluation of effects on LRP5/6 phosphorylation

  • Analysis of β-catenin stabilization and nuclear translocation

• CAPRIN2 manipulation controls:

  • Multiple siRNA/shRNA constructs targeting different regions

  • Rescue experiments with RNAi-resistant CAPRIN2 constructs

  • CAPRIN1 overexpression to confirm specificity of CAPRIN2 effects

These controls will help establish the specific role of CAPRIN2 in facilitating LRP5/6 phosphorylation by GSK3 and enhancing Axin-LRP5/6 interactions in the Wnt signaling cascade .

What are the critical parameters for optimizing western blot analysis with CAPRIN2 antibodies?

For successful western blot detection of CAPRIN2:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation-dependent interactions

  • Recommended positive control samples: Y79 cells, SH-SY5Y cells, HEK-293 cells

• Gel electrophoresis conditions:

  • Use 6-8% gels due to the large size of CAPRIN2 (126-150 kDa)

  • Longer running times may be needed for adequate separation

  • Consider gradient gels for better resolution

• Transfer conditions:

  • Use wet transfer methods for large proteins

  • Extend transfer time (overnight at lower voltage is often optimal)

  • Consider adding SDS to transfer buffer (0.1%) to facilitate large protein transfer

• Blocking and antibody incubation:

  • Recommended dilution: 1:500-1:1000 for primary antibody

  • BSA-based blocking may be preferable to milk for phospho-specific applications

  • Longer primary antibody incubation (overnight at 4°C) often yields better results

• Detection optimization:

  • For HRP-conjugated antibodies, use enhanced chemiluminescence substrate appropriate for the expected protein abundance

  • Longer exposure times may be necessary for detecting endogenous CAPRIN2 in some tissues

Following these parameters should result in specific detection of CAPRIN2 at the expected molecular weight range of 126-150 kDa .

How should I validate knockdown or knockout models of CAPRIN2 for functional studies?

Comprehensive validation of CAPRIN2 manipulation requires multiple approaches:

• mRNA level validation:

  • Quantitative RT-PCR with primers targeting different exons

  • Analysis of potential compensatory changes in related genes (e.g., CAPRIN1)

  • Verification that knockdown targets all relevant splice variants

• Protein level validation:

  • Western blot analysis using antibodies targeting different epitopes

  • Immunofluorescence to confirm reduction in the appropriate cellular compartments

  • Quantification of knockdown efficiency (typically aim for >70% reduction)

• Functional validation:

  • Confirmation of altered downstream pathway activity:

    • For Wnt signaling: LRP5/6 phosphorylation, β-catenin accumulation

    • For AVP regulation: changes in mRNA poly(A) tail length, AVP mRNA levels

  • Rescue experiments with wild-type CAPRIN2 to confirm specificity

  • Analysis of physiological endpoints (e.g., fluid homeostasis parameters for hypothalamic studies)

• Off-target effect assessment:

  • Use multiple independent shRNA/siRNA sequences

  • Include scrambled sequence controls

  • For CRISPR/Cas9 approaches, verify no modifications at predicted off-target sites

In studies of CAPRIN2 in the hypothalamus, successful validation included verification of significant reduction in both mRNA and protein levels, along with functional changes in AVP regulation and physiological parameters of water balance .

How can I investigate CAPRIN2's role in mRNA polyadenylation versus deadenylation processes?

To dissect CAPRIN2's specific effects on poly(A) tail dynamics:

• Poly(A) tail length measurement techniques:

  • Northern blot analysis:

    • Compare RNA size before and after oligo(dT)/RNase H treatment

    • Quantify the size difference representing the poly(A) tail

    • This approach has shown that CAPRIN2 knockdown decreases AVP mRNA length in a poly(A)-dependent manner

  • ePAT (extension Poly(A) Test) or LM-PAT:

    • More sensitive method for poly(A) tail length determination

    • Can detect changes in specific transcripts from limited samples

  • TAIL-seq or PAL-seq:

    • Genome-wide approaches to identify all CAPRIN2-regulated poly(A) tails

    • Can help identify common motifs in regulated transcripts

• Mechanistic investigations:

  • RNA-protein complex immunoprecipitation:

    • Determine if CAPRIN2 associates with polyadenylation machinery components

    • Investigate interactions with deadenylase complexes

    • Analyze how these interactions change under osmotic stress conditions

  • In vitro polyadenylation assays:

    • Use recombinant proteins and in vitro transcribed RNAs

    • Test direct effects on poly(A) polymerase activity

    • Assess competition with deadenylase complexes

• Regulatory element identification:

  • Mutational analysis of AVP mRNA 3'UTR to identify CAPRIN2-responsive elements

  • CLIP-seq (crosslinking immunoprecipitation-sequencing) to map CAPRIN2 binding sites

  • Motif analysis across CAPRIN2-regulated transcripts

Research has noted that it remains unclear whether CAPRIN2 promotes polyadenylation or prevents deadenylation of AVP mRNA, suggesting both mechanisms should be investigated .

What technical approaches can determine if CAPRIN2 homodimerization is required for its RNA-binding function?

Investigating the relationship between CAPRIN2 dimerization and RNA binding requires multiple complementary approaches:

• Structure-function analysis:

  • Generate mutations disrupting the dimerization interface in the HR1 domain

  • Crystal structure information shows CAPRIN2's HR1 domain forms a homodimer resembling a pair of scissors

  • Test mutants for both dimerization ability and RNA binding capacity

• Dimerization assessment techniques:

  • Co-immunoprecipitation: Using differently tagged versions of CAPRIN2

  • FRET or BiFC analysis: For monitoring dimerization in living cells

  • Size exclusion chromatography: To separate monomeric and dimeric forms

  • Analytical ultracentrifugation: For precise determination of oligomeric states

• RNA binding evaluation methods:

  • RNA immunoprecipitation (RIP) with wild-type and dimerization-defective mutants

  • Electrophoretic mobility shift assays (EMSA) with recombinant proteins

  • Surface plasmon resonance to measure binding kinetics and affinity

  • RNA pull-down assays followed by western blotting for CAPRIN2

• Functional consequence analysis:

  • Test dimerization mutants for ability to regulate AVP mRNA poly(A) tail length

  • Assess effects on AVP mRNA stability

  • Evaluate impact on AVP production and secretion in cellular models

  • For Wnt signaling, determine effects on LRP5/6 phosphorylation

Understanding the relationship between dimerization and RNA binding would provide important insights into CAPRIN2's molecular mechanism of action and could identify novel therapeutic targets for disorders of water balance or Wnt signaling dysregulation .

How can I design experiments to identify the complete repertoire of CAPRIN2-bound mRNAs in different physiological states?

To comprehensively identify CAPRIN2's mRNA targets across different conditions:

• CLIP-seq approaches:

  • HITS-CLIP or PAR-CLIP:

    • Crosslink RNA-protein complexes in vivo

    • Immunoprecipitate CAPRIN2 under stringent conditions

    • Extract, sequence, and map bound RNAs

    • Compare binding patterns between normal and osmotically challenged states

  • iCLIP or eCLIP:

    • Provides single-nucleotide resolution of binding sites

    • Can identify exact motifs recognized by CAPRIN2

• RIP-seq methodology:

  • Perform RNA immunoprecipitation without crosslinking

  • Deep sequence associated RNAs

  • Compare with appropriate IgG controls

  • Analyze in multiple physiological conditions (e.g., euhydrated vs. dehydrated or salt-loaded)

• RNA motif identification:

  • Computational analysis of binding sites to identify consensus sequences

  • Validate motifs using reporter assays with wild-type and mutated binding sites

  • Test if identified motifs are sufficient to confer CAPRIN2 regulation

• Integration with functional outcomes:

  • Correlate binding with changes in:

    • mRNA stability

    • Poly(A) tail length

    • Translational efficiency

  • Perform pathway enrichment analysis of target mRNAs

  • Validate key targets with individual assays

• Tissue-specific considerations:

  • Compare CAPRIN2 RNA targets between different tissues (e.g., hypothalamus vs. eye)

  • Analyze cell type-specific binding (e.g., neurons vs. lens cells)

  • Consider developmental stage differences in binding profiles

This comprehensive approach would extend current knowledge beyond the established AVP mRNA target to identify the full spectrum of CAPRIN2-regulated transcripts, potentially revealing novel physiological roles and regulatory mechanisms .