RPAIN Antibody

What is RPAIN and what is its primary function in cellular processes?

RPAIN (RPA Interacting Protein, also known as RIP) serves as a specialized nuclear transporter of the Replication Protein A (RPA) complex. Unlike conventional nuclear import mediated by importin proteins, RPAIN functions as an adapter protein that facilitates the import of the RPA complex into the nucleus, likely through interactions with importin beta . The RPA complex itself is a critical single-stranded DNA binding protein involved in numerous eukaryotic DNA processes including replication, repair, and recombination .

RPAIN exists in multiple isoforms with varying cellular localizations and functions:

• The longer isoform (hRIPα) primarily localizes to the cytoplasm and shuttles between cytoplasm and nucleus to transport RPA

• The shorter isoform (hRIPβ) is sumoylated and localizes specifically to promyelocytic leukemia (PML) nuclear bodies, potentially mediating the localization of the RPA complex into these structures

This strategic localization suggests RPAIN plays a critical role in DNA metabolism by ensuring RPA is available where needed within the nuclear compartment.

What are the key molecular characteristics of RPAIN protein?

RPAIN exhibits several important molecular characteristics relevant to research applications:

The discrepancy between calculated and observed molecular weights likely reflects post-translational modifications such as sumoylation in certain isoforms, particularly in isoform 2 localized to PML nuclear bodies .

What types of RPAIN antibodies are commercially available for research use?

Several validated RPAIN antibodies are available for researchers, including both polyclonal and monoclonal options:

When selecting an antibody, consider the specific experimental application, required species reactivity, and whether polyclonal (offering broader epitope recognition) or monoclonal (offering higher specificity) characteristics better suit your research needs .

What are the optimal protocols for using RPAIN antibodies in Western blot applications?

For successful Western blot detection of RPAIN, consider the following optimized protocol parameters:

Methodological recommendations:

• Protein extraction should include protease inhibitors to prevent degradation

• Include phosphatase inhibitors if investigating phosphorylated forms of RPAIN

• For detecting multiple isoforms, use gradient gels (4-20%) for better resolution

• Consider using RIPA buffer for enhanced extraction efficiency

• When analyzing nuclear vs. cytoplasmic distribution of RPAIN isoforms, perform subcellular fractionation prior to Western blot

• Use appropriate positive controls (A375 or Jurkat cells are recommended)

Remember that the observed molecular weight may vary depending on post-translational modifications and the specific isoform being detected. The publication with PMID: 16135809 reported molecular weights of 30 and 45 kDa, which may represent different isoforms or modified versions of RPAIN .

How should RPAIN antibodies be optimized for immunohistochemistry applications?

For successful IHC applications with RPAIN antibodies, follow these research-validated protocols:

For polyclonal antibody 15220-1-AP:

• Recommended dilution: 1:20-1:200

• Antigen retrieval: Use TE buffer pH 9.0 (primary recommendation) or citrate buffer pH 6.0

• Verified positive tissues: Human ovary tumor tissue

• Consider testing multiple concentrations as optimal dilution may be sample-dependent

For polyclonal antibody A11439:

• Recommended starting concentration: 2.5 μg/mL

• Verified positive tissues: Mouse stomach tissue

General methodological considerations:

• Include appropriate positive and negative controls (human ovary tumor tissue is recommended as a positive control)

• For formalin-fixed paraffin-embedded (FFPE) tissues, ensure complete deparaffinization

• Include blocking steps to reduce nonspecific binding

• Consider the use of amplification systems for detecting low-abundance targets

• For dual staining to differentiate isoforms, test antibodies raised in different host species

Optimizing incubation time and temperature may be necessary for specific tissue types. Always validate new tissue types with known positive controls before proceeding with experimental samples .

What is the optimal approach for immunoprecipitation using RPAIN antibodies?

For successful immunoprecipitation (IP) of RPAIN:

• Antibody recommendation: Use polyclonal antibody 15220-1-AP at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate

• Verified positive sample: A375 cells have been confirmed for successful IP applications

• Protocol considerations:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Form antibody-protein complexes by overnight incubation at 4°C

  • Wash stringently to remove non-specific interactions

  • For detecting RPAIN interactions with RPA complex components, consider crosslinking before lysis

  • For studying sumoylated isoforms, include N-ethylmaleimide in lysis buffers to inhibit SUMO proteases

IP experiments can be particularly valuable for studying RPAIN's interactions with the RPA complex and other proteins involved in DNA replication and repair pathways, as well as for identifying post-translational modifications on different RPAIN isoforms .

How can researchers differentiate between RPAIN isoforms in experimental systems?

Distinguishing between RPAIN isoforms requires strategic experimental approaches:

Western blot differentiation:

• Use gradient gels (4-20%) for optimal separation of different molecular weight isoforms

• The longer isoform (hRIPα) typically appears at 25-27 kDa

• The shorter isoforms appear at 16-19 kDa and 12 kDa

• Sumoylated isoform 2 (hRIPβ) may appear at higher molecular weights (30-45 kDa)

Subcellular localization:

• Perform cellular fractionation to separate cytoplasmic and nuclear components

• The longer isoform (hRIPα) should predominantly appear in cytoplasmic fractions

• The sumoylated isoform 2 (hRIPβ) should predominantly localize to nuclear fractions

Immunofluorescence approach:

• Co-stain with PML nuclear body markers to identify isoform 2

• Use antibodies that can detect different epitopes specific to each isoform

• Consider using overexpression systems with tagged versions of specific isoforms as positive controls

When interpreting results, remember that the relative abundance of isoforms may vary between different cell types and under different cellular conditions (e.g., cell cycle phase, DNA damage response) .

What are the key considerations when studying RPAIN's role in the DNA damage response?

When investigating RPAIN's function in DNA damage response pathways:

How can researchers troubleshoot inconsistent RPAIN antibody performance across different experimental systems?

When encountering variable results with RPAIN antibodies:

• Sample preparation issues:

  • RPAIN isoforms may degrade quickly; use fresh samples and include protease inhibitors

  • For nuclear isoforms, ensure proper subcellular fractionation techniques

  • Consider the expression level of RPAIN in your specific cell line/tissue (may require optimization of loading amounts)

• Antibody selection considerations:

  • Different antibodies may preferentially recognize specific isoforms or epitopes

  • Solution: Use multiple antibodies targeting different regions of RPAIN

  • Monoclonal antibodies (like 68546-1-Ig) offer high specificity but might miss some isoforms

  • Polyclonal antibodies (like 15220-1-AP) recognize multiple epitopes but may show more background

• Protocol optimization:

  • For Western blot: Adjust transfer conditions for different molecular weight isoforms

  • For IHC: Test multiple antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • For low abundance: Consider using signal amplification systems

• Validation approaches:

  • Use positive control samples with known RPAIN expression (A375, Jurkat cells)

  • Consider using overexpression systems as positive controls

  • For knockout validation, use CRISPR-Cas9 generated RPAIN-null cells

  • Peptide competition assays can confirm antibody specificity

What experimental design is optimal for studying RPAIN-mediated RPA nuclear import?

To effectively study RPAIN's role in RPA nuclear transport:

• Recommended cellular systems:

  • Cell lines with well-characterized nuclear import mechanisms (U2OS, HeLa)

  • Cell types that undergo extensive DNA replication and repair (proliferating cells)

• Methodological approaches:

  • Fluorescence microscopy with tagged RPA subunits to track localization

  • RPAIN knockdown/knockout to assess impact on RPA nuclear import

  • Complementation with different RPAIN isoforms to determine isoform-specific functions

  • Co-immunoprecipitation to detect RPAIN-importin β and RPAIN-RPA interactions

  • In vitro nuclear import assays using permeabilized cells

• Key experimental controls:

  • General import inhibitors (e.g., wheat germ agglutinin) as positive controls for import blockade

  • Importin β knockdown to distinguish RPAIN-specific vs. general import defects

  • Cell cycle synchronization to normalize for cell cycle effects on nuclear import

• Advanced approaches:

  • FRAP (Fluorescence Recovery After Photobleaching) to measure import kinetics

  • Proximity ligation assays to detect RPAIN-RPA interactions in situ

  • Structure-function analysis using RPAIN mutants lacking specific interaction domains

  • Mass spectrometry to identify post-translational modifications regulating RPAIN transport activity

How should researchers interpret discrepancies in observed molecular weights for RPAIN across different studies?

When encountering variation in RPAIN molecular weight observations:

• Source of variability:

  • Multiple isoforms with different base molecular weights (25-27 kDa, 16-19 kDa, 12 kDa)

  • Post-translational modifications, particularly sumoylation of isoform 2

  • Different gel systems and protein standards can affect apparent molecular weight

  • Publication PMID:16135809 reported molecular weights of 30 and 45 kDa

• Analytical approach:

  • Compare observed weights with calculated weights (25 kDa theoretical)

  • Consider running isoform-specific recombinant standards

  • Use phosphatase/desumoylase treatment to identify contribution of modifications

  • Verify with mass spectrometry for absolute molecular weight determination

• Experimental validation:

  • Use multiple antibodies targeting different epitopes

  • Perform isoform-specific knockdown to identify specific bands

  • Consider 2D gel electrophoresis to separate based on both size and charge

  • For suspected sumoylation, perform immunoprecipitation followed by SUMO-specific Western blot

What statistical approaches are most appropriate for quantifying RPAIN expression changes in response to experimental conditions?

For robust quantification of RPAIN expression changes:

• Recommended quantification methods:

  • Densitometry analysis of Western blots (normalize to stable housekeeping proteins)

  • qRT-PCR for mRNA level changes (use multiple reference genes)

  • Quantitative immunofluorescence (measure mean fluorescence intensity)

• Statistical approaches:

  • For comparing two conditions: Student's t-test (paired when appropriate)

  • For multiple conditions: ANOVA with appropriate post-hoc tests

  • For non-normally distributed data: Non-parametric tests like Mann-Whitney U or Kruskal-Wallis

  • Include power analysis to determine appropriate sample size

• Experimental design considerations:

  • Perform at least three biological replicates

  • Include technical replicates within each biological replicate

  • Use blinding procedures when possible to prevent bias

  • Consider the use of positive controls (known inducers of RPAIN expression changes)

• Addressing common analytical challenges:

  • When multiple isoforms are present, analyze each band separately

  • For comparing across different cell types, consider relative fold changes rather than absolute values

  • Account for differences in antibody affinity when comparing different RPAIN isoforms

  • Consider normalization to total protein loading (e.g., using stain-free technology) rather than single housekeeping proteins

How can researchers design antibody-based experiments to study RPAIN interactions with the DNA repair machinery?

To effectively investigate RPAIN's role in DNA repair pathways:

• Experimental design strategies:

  • Co-immunoprecipitation of RPAIN with RPA and other repair factors

  • Proximity ligation assay (PLA) to detect protein-protein interactions in situ

  • Chromatin immunoprecipitation (ChIP) to detect RPAIN recruitment to damaged DNA

  • FRET/BRET approaches for real-time interaction monitoring

• DNA damage induction methods:

  • UV irradiation (global genomic nucleotide excision repair)

  • Micro-irradiation with laser (localized damage for live imaging)

  • Chemical agents (cisplatin, MMS, hydroxyurea) for specific damage types

  • Consider time-course experiments to capture transient interactions

• Critical controls:

  • Include both damaged and undamaged conditions

  • Use RPA subunit knockdown to confirm specificity of interactions

  • Include known RPA interactors as positive controls

  • For protein-protein interactions, consider reciprocal co-IP experiments

• Advanced analytical approaches:

  • Mass spectrometry of RPAIN interactome before and after DNA damage

  • Quantitative analysis of co-localization with repair factors at damage sites

  • Single-molecule imaging to track RPAIN dynamics at repair sites

  • Consider cell cycle synchronization as repair pathway activity varies with cell cycle phase

What are promising approaches for developing isoform-specific RPAIN antibodies for research applications?

For developing next-generation isoform-specific RPAIN antibodies:

• Epitope selection strategies:

  • Target unique sequence regions specific to each isoform

  • For hRIPα (cytoplasmic isoform), target unique C-terminal sequences

  • For hRIPβ (nuclear isoform), target regions containing or adjacent to sumoylation sites

  • Consider generating phospho-specific antibodies if isoforms differ in phosphorylation status

• Production approaches:

  • Recombinant antibody technology for higher specificity

  • Phage display screening against specific isoforms

  • Consider nanobodies for accessing sterically hindered epitopes

  • Monoclonal antibody development using isoform-specific immunogens

• Validation requirements:

  • Expression systems overexpressing single isoforms as positive controls

  • Isoform-specific knockdowns as negative controls

  • Mass spectrometry confirmation of isoform-specific immunoprecipitation

  • Cross-validation with existing antibodies and orthogonal methods

• Application-specific considerations:

  • For live-cell imaging, develop cell-permeable nanobodies

  • For super-resolution microscopy, consider site-specific labeling approaches

  • For multiplexed detection, generate antibodies from different host species

  • For quantitative applications, consider generating recombinant antibodies with defined affinity characteristics

How might CRISPR-based approaches enhance our understanding of RPAIN function in DNA metabolism?

CRISPR-Cas technologies offer powerful approaches for RPAIN functional studies:

• Genome editing applications:

  • CRISPR knockout of RPAIN to assess global effects on RPA localization and function

  • Isoform-specific editing by targeting isoform-unique exons

  • Knock-in of tags (GFP, FLAG) at endogenous loci for physiological expression levels

  • Introduction of point mutations to disrupt specific functional domains or PTM sites

• CRISPR screening approaches:

  • Genome-wide CRISPR screens for synthetic lethality with RPAIN deficiency

  • Targeted screens of DNA repair genes to identify genetic interactions

  • CRISPRi/CRISPRa for reversible modulation of RPAIN expression

  • Domain-focused saturation mutagenesis to map functional regions

• CRISPR imaging applications:

  • CRISPR-based tagging for live-cell tracking of endogenous RPAIN

  • Simultaneous imaging of RPAIN and RPA during DNA damage response

  • Optogenetic control of RPAIN function to probe temporal aspects

• Experimental design considerations:

  • Include appropriate controls (non-targeting gRNAs)

  • Validate editing efficiency through sequencing and protein expression analysis

  • Consider potential compensation by related proteins

  • For essential functions, use inducible or partial knockdown approaches

What are emerging methods for studying the dynamic interplay between RPAIN isoforms and the sumoylation machinery?

To investigate the complex relationship between RPAIN isoforms and sumoylation:

• Advanced biochemical approaches:

  • In vitro sumoylation assays with recombinant RPAIN isoforms

  • Proximity-dependent labeling (BioID, APEX) to identify nearby sumoylation machinery

  • Quantitative proteomics to measure sumoylation stoichiometry

  • SUMO-specific protein microarrays to profile isoform-specific interactions

• Cellular imaging strategies:

  • FRET-based sensors for detecting sumoylation in real-time

  • Split fluorescent protein complementation to visualize RPAIN-SUMO interaction

  • Multi-color live-cell imaging to track RPAIN, SUMO, and PML dynamics

  • Super-resolution microscopy to resolve subnuclear structures

• Genetic manipulation approaches:

  • CRISPR-based editing of sumoylation sites in RPAIN

  • Inducible expression of SUMO proteases to rapidly desumoylate targets

  • Targeted degradation of sumoylated proteins using engineered SUMO-targeted ubiquitin ligases

  • Expression of SUMO-trapped mutants to stabilize transient interactions

• Mathematical modeling:

  • Kinetic modeling of the sumoylation/desumoylation cycle

  • Agent-based models of nuclear body formation and dynamics

  • Systems biology approaches to integrate multiple datasets

  • Machine learning to identify patterns in complex spatiotemporal datasets

These emerging approaches provide powerful tools for dissecting the complex relationship between RPAIN isoforms, sumoylation, and nuclear organization in DNA metabolism .

What are the most reliable positive control samples for validating RPAIN antibody specificity?

Based on validated experimental data, the following samples serve as reliable positive controls:

Methodological recommendations:

• Include both positive and negative control samples in each experiment

• For knockout validation, consider using RPAIN CRISPR knockout cell lines

• For overexpression controls, use cells transfected with RPAIN expression constructs

• When possible, use multiple cell lines to confirm antibody specificity across different cellular contexts

• Include loading controls and normalize to total protein for quantitative applications

What are the optimal storage and handling conditions to maintain RPAIN antibody performance?

To ensure maximum stability and performance of RPAIN antibodies:

• Storage recommendations:

  • Store antibodies at -20°C for long-term stability (up to one year)

  • For short-term storage (up to three months), 4°C is acceptable for some formulations

  • Avoid repeated freeze-thaw cycles that can degrade antibody quality

  • For 15220-1-AP and 68546-1-Ig: Store in PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Aliquoting best practices:

  • Divide antibodies into single-use aliquots upon receipt

  • Use sterile tubes and conditions when preparing aliquots

  • Record date of aliquoting and track usage of each aliquot

  • For small volume antibodies (20μl), aliquoting may be unnecessary for -20°C storage

• Working dilution preparation:

  • Prepare fresh working dilutions for each experiment

  • Use high-quality, freshly prepared buffers

  • For Western blot applications, consider using milk or BSA as blocking agents

  • Filter solutions if precipitation is observed

• Quality control measures:

  • Include positive controls with each new experiment or antibody lot

  • Monitor signal-to-noise ratio as an indicator of antibody performance

  • Consider batch testing when receiving new antibody lots

  • Document lot numbers and correlate with experimental outcomes

How can researchers effectively design experiments to address contradictory findings in the RPAIN literature?

When confronting contradictory results in RPAIN research:

• Systematic approach to literature discrepancies:

  • Identify specific points of contradiction (localization, function, interactions)

  • Analyze methodological differences that might explain discrepancies

  • Consider cell type-specific or context-dependent effects

  • Evaluate reagent differences (antibodies, expression constructs)

• Experimental design strategies:

  • Use multiple, complementary techniques to address the same question

  • Include appropriate positive and negative controls for each method

  • Consider both gain-of-function and loss-of-function approaches

  • Validate key findings in multiple cell types or experimental systems

• Critical controls:

  • For antibody-based discrepancies: Use multiple antibodies targeting different epitopes

  • For functional studies: Include rescue experiments with wild-type and mutant constructs

  • For localization discrepancies: Use both biochemical fractionation and imaging approaches

  • For interaction studies: Perform both endogenous and overexpression analyses

• Collaborative and reproducibility approaches:

  • Consider blind analysis of data to minimize bias

  • Implement rigorous statistical analyses with appropriate sample sizes

  • Share reagents and protocols with collaborators for independent validation

  • Consider pre-registering experimental designs for key validations