CLPS3 Antibody

What is CLPS3 and why is it important in research?

CLPS3 is an Arabidopsis protein orthologous to human polyadenylation factors that plays a crucial role in RNA processing. Research has demonstrated that CLPS3 forms complexes with other polyadenylation factors including FY, CPSF100, and CPSF160 . These interactions are vital for understanding RNA processing mechanisms in plants. As a component of the cleavage and polyadenylation machinery, CLPS3 contributes to gene expression regulation through alternative polyadenylation, affecting developmental processes and responses to environmental stimuli. Antibodies against CLPS3 enable researchers to track its expression, localization, and interactions within cellular contexts, providing insights into fundamental biological processes.

How does CLPS3 function in the polyadenylation complex?

CLPS3 participates in RNA 3' end processing by interacting with multiple components of the polyadenylation machinery. Tandem affinity purification experiments have revealed that CLPS3 forms complexes with FY (the plant ortholog of WDR33), CPSF100, and CPSF160 . Notably, CLPS3 directly interacts with PCFS4 and forms in vivo complexes with FY and additional unidentified proteins. These interactions suggest CLPS3 functions as a scaffold or regulatory component within the larger polyadenylation complex, potentially modulating substrate recognition or enzymatic activity. The polyadenylation complex influences gene expression patterns by determining the site of polyadenylation, which can affect mRNA stability, translation efficiency, and protein isoform production.

What are the recommended methods for validating CLPS3 antibody specificity?

Antibody validation is critical for ensuring experimental reliability. For CLPS3 antibodies, a comprehensive validation approach should include:

• Western blot analysis: Using wild-type and CLPS3 knockout/knockdown samples to confirm antibody specificity

• Immunoprecipitation: Verifying that the antibody can pull down CLPS3 and known interacting partners

• Peptide competition assay: Demonstrating signal reduction when antibody is pre-incubated with purified CLPS3 peptide

• Immunocytochemistry correlation: Comparing antibody signal with GFP-tagged CLPS3 localization patterns

• Cross-reactivity testing: Evaluating potential cross-reactivity with related proteins such as other CLP family members

Drawing from antibody validation methodologies in similar systems, comprehensive characterization should include specificity, sensitivity, and reproducibility assessment across multiple techniques .

How can CLPS3 antibodies be used in ChIP-Seq experiments to study RNA processing?

ChIP-Seq experiments with CLPS3 antibodies can reveal genome-wide binding patterns, illuminating its role in RNA processing. Based on related studies, the following methodology is recommended:

• Cross-linking: Formaldehyde fixation of plant tissue or cultured cells (1% for 10 minutes at room temperature)

• Chromatin preparation: Sonication to achieve DNA fragments of 200-500bp

• Immunoprecipitation: Using validated CLPS3 antibodies with appropriate controls (IgG, input)

• DNA purification and library preparation: Standard NGS library protocols

• Data analysis: Alignment to reference genome and peak calling

Similar ChIP-Seq approaches with RNA processing factors have revealed co-localization patterns with RNA polymerase II phosphorylated at Ser2, particularly at 3' ends of genes . For CLPS3, researchers should analyze binding patterns around polyadenylation sites and correlate with RNA-Seq data to identify regulated transcripts. This approach can identify novel targets and context-specific regulation mechanisms.

What are the optimal conditions for immunoprecipitation experiments using CLPS3 antibodies?

Successful immunoprecipitation of CLPS3 and its associated complexes requires carefully optimized conditions:

• Lysis buffer composition: 50mM Tris-HCl (pH 7.5), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease inhibitors

• Cross-linking considerations: For transient interactions, consider mild formaldehyde (0.1-0.3%) or DSP crosslinking

• Antibody amount: Typically 2-5μg per mg of total protein extract

• Incubation parameters: Overnight at 4°C with gentle rotation

• Washing stringency: Multiple washes with decreasing salt concentration to remove non-specific interactions

Drawing from the CLPS3-TAP purification methodology, whole cell extracts should be prepared and subjected to affinity purification under conditions that maintain complex integrity . Western blotting for known interacting partners (FY, CPSF100, CPSF160) serves as positive controls to verify successful immunoprecipitation.

How can researchers generate and validate monoclonal antibodies against CLPS3?

Generating high-quality monoclonal antibodies against CLPS3 involves several critical steps:

• Antigen design: Choose unique epitopes with high antigenicity and low homology to related proteins

• Immunization strategy: Utilize CLPS3 knockout mice immunized with human/target CLPS3 mRNA lipid nanoparticles with adjuvants like AddaVax

• Hybridoma screening: Select clones based on binding specificity to CLPS3-overexpressing cells versus controls

• Antibody purification: Standard protein A/G methods followed by characterization

• Validation tests: Cross-reactivity testing, epitope mapping, and functional assays

This approach mirrors successful antibody generation strategies used for other proteins like EpCAM and CLDN3 . After generating hybridomas, RNA extraction and cDNA synthesis enable sequence determination and subsequent humanization if desired. Validation should include binding assays with wild-type versus CLPS3-overexpressing cells, as well as knockout controls.

How can CLPS3 antibodies help elucidate its role in alternative polyadenylation?

CLPS3 antibodies are invaluable tools for understanding alternative polyadenylation mechanisms:

• RNA immunoprecipitation (RIP): Identify direct RNA targets of CLPS3 to map binding sites relative to polyadenylation sites

• CLIP-Seq: Provide single-nucleotide resolution of CLPS3-RNA interactions in vivo

• Functional validation: Compare polyadenylation patterns in wild-type versus CLPS3-depleted samples

• Protein complex identification: Use antibodies to pull down and characterize components of CLPS3-containing complexes

Research on related proteins suggests that CLPS3 may influence alternative polyadenylation of genes such as FLC, which is regulated by long non-coding antisense RNAs that undergo alternative polyadenylation . Metagene analysis incorporating CLPS3 ChIP-Seq data with RNA-Seq and poly(A)-seq could reveal preferential association with specific polyadenylation signals or RNA motifs, enabling construction of a comprehensive model of CLPS3's role in transcript processing.

What are the common technical challenges when working with CLPS3 antibodies in plant tissue samples?

Plant tissues present unique challenges for antibody-based experiments:

When working with Arabidopsis samples, the affinity purification approach used for CLPS3-TAP can inform troubleshooting strategies . Different extraction buffers may be required for various tissue types, and the inclusion of plant-specific protease inhibitors is essential. Additionally, optimizing antibody concentrations through titration experiments can help maximize signal-to-noise ratios.

How can researchers use CLPS3 antibodies to investigate its potential interactome?

Comprehensive interactome analysis of CLPS3 can be achieved through several antibody-dependent approaches:

• Co-immunoprecipitation with mass spectrometry: Pull down CLPS3 complexes and identify interacting partners through MS/MS analysis

• Proximity labeling: Combine CLPS3 antibodies with BioID or APEX2 proximity labeling to identify proteins in close spatial proximity

• Immunofluorescence co-localization: Visualize potential interactions through co-localization studies with other polyadenylation factors

• Split-reporter validation: Confirm direct interactions identified through antibody-based methods

Drawing on methods used for analyzing the ANK domain interactome of human CLPB , researchers can implement stringent enrichment criteria (fold-change > 4 and p-value < 0.01) when analyzing mass spectrometry data from CLPS3 immunoprecipitation. The previously identified interactions with FY, CPSF100, and CPSF160 serve as positive controls . Researchers should pay particular attention to proteins involved in RNA processing, transcription termination, and alternative splicing to build a comprehensive model of CLPS3's functional network.

How do antibodies against CLPS3 compare with those targeting its human orthologs?

Comparing plant CLPS3 antibodies with those targeting human orthologs reveals important considerations:

• Epitope conservation: Analysis of sequence homology between plant CLPS3 and human orthologs determines potential cross-reactivity

• Functional domain recognition: Antibodies targeting conserved functional domains may show cross-species reactivity

• Application versatility: Human ortholog antibodies often have broader validation across multiple techniques

• Detection sensitivity: Species-specific optimization may be required for optimal signal-to-noise ratios

The human CLPB protein shares functional similarities with plant CLPS3, though they function in different cellular compartments with distinct binding partners . When developing cross-species reactive antibodies, researchers should target highly conserved epitopes while avoiding regions with significant divergence. Validation across multiple species requires comprehensive testing in relevant cellular contexts for each target organism.

Can CLPS3 antibodies provide insights into disease mechanisms related to RNA processing defects?

While CLPS3 is a plant protein, studying its function can inform understanding of conserved RNA processing mechanisms relevant to human disease:

• Comparative studies: Using antibodies against both plant CLPS3 and human orthologs to identify conserved and divergent functions

• Disease model systems: Applying insights from plant CLPS3 studies to human cellular models of RNA processing disorders

• Therapeutic target identification: Leveraging conserved mechanisms to develop novel approaches to RNA processing diseases

Research on human CLPB (SKD3) demonstrates that dysfunction through mutations is associated with diseases like 3-methylglutaconic aciduria and severe congenital neutropenia . By understanding fundamental mechanisms of RNA processing through CLPS3 studies, researchers can potentially identify therapeutic targets for human diseases involving related pathways. This translational approach underscores the value of basic research in model organisms for advancing human health.

How can CLPS3 antibodies help characterize its role in transcription termination?

Recent research suggests connections between polyadenylation factors and transcription termination:

• ChIP-Seq analysis: Map CLPS3 binding relative to transcription termination sites genome-wide

• Sequential ChIP: Determine co-occupancy with RNA polymerase II and termination factors

• Nascent RNA analysis: Combine CLPS3 immunoprecipitation with nascent RNA sequencing

• Termination assays: Measure readthrough transcription in CLPS3-depleted versus control samples

Research on FPA, another RNA processing factor in Arabidopsis, revealed co-localization with Ser2 phosphorylated RNA Polymerase II at 3' ends of genes, suggesting involvement in transcription termination . Similar approaches could elucidate CLPS3's potential role in this process. Researchers should analyze CLPS3 binding patterns in relation to premature termination sites and gene boundaries to understand its contribution to defining transcript 3' ends.

How can CLPS3 antibodies be integrated with single-cell technologies for RNA processing studies?

Integrating CLPS3 antibodies with single-cell technologies offers exciting research opportunities:

• Single-cell ChIP-Seq: Map CLPS3 chromatin occupancy in individual cells to detect cell-type-specific patterns

• CUT&Tag in single cells: Profile CLPS3 binding with higher sensitivity and lower background

• Spatial transcriptomics: Combine tissue localization of CLPS3 with transcript maps

• Multi-omics integration: Correlate CLPS3 binding with single-cell transcriptomes and alternative polyadenylation events

These approaches can reveal cell-type-specific roles of CLPS3 in RNA processing, potentially identifying specialized functions in different tissues or developmental stages. For example, CLPS3 might preferentially regulate certain subsets of transcripts in specific cell types, contributing to cell identity and function. Methodological adaptations for plant single-cell studies will require optimization of tissue dissociation protocols and antibody penetration.

What are the considerations for developing phospho-specific antibodies against CLPS3?

Phosphorylation can regulate protein activity, interactions, and localization. For CLPS3 phospho-specific antibodies:

• Phosphorylation site prediction: Identify potential sites through computational analysis and proteomic data

• Antigen design: Synthesize phosphopeptides corresponding to predicted sites

• Screening strategy: Test antibodies against phosphorylated versus non-phosphorylated peptides

• Validation approach: Confirm specificity using phosphatase treatment and phosphomimetic mutants

• Functional correlation: Link specific phosphorylation events to CLPS3 activity or interactions

When developing phospho-specific antibodies, researchers must ensure that the antibody recognizes the phosphorylated residue in the context of the surrounding protein sequence. Validation should include treatment with phosphatases to demonstrate phospho-specificity. Additionally, mutational analysis of predicted phosphorylation sites can provide functional validation of antibody specificity and biological significance.

How can structural information enhance CLPS3 antibody design and experimental applications?

Structural insights can significantly improve antibody design and experimental strategies:

• Epitope accessibility: Target regions exposed in native protein conformation

• Functional domain targeting: Design antibodies against specific functional domains for mechanistic studies

• Conformation-specific antibodies: Develop antibodies recognizing particular structural states of CLPS3

• Complex-disrupting antibodies: Create tools that specifically interfere with certain protein-protein interactions

Drawing on structural characterization methods used for human CLPB , researchers could employ cryo-EM or X-ray crystallography to determine CLPS3 structure in different functional states. This information would enable rational antibody design targeting specific epitopes relevant to function. For example, antibodies targeting the interface between CLPS3 and its binding partners could serve as valuable tools for dissecting complex assembly and function in vitro and in vivo.