prrc1 Antibody

What is PRRC1 and why is it relevant for research?

PRRC1 (Proline-Rich Coiled-Coil 1) is a protein-coding gene located on chromosome 5q23.2. It contains two distinct structural domains: a proline-rich region at the N-terminus and a DUF84 (Domain of Unknown Function 84) region at the C-terminus. The protein encoded by this gene consists of 445 amino acids distributed across 6 exons, with a predicted molecular weight of 46.7 kDa and an isoelectric point of 5.46. PRRC1 is evolutionarily conserved across multiple species including chimpanzee, rhesus monkey, dog, cow, mouse, rat, chicken, zebrafish, and frog, suggesting important biological functions. It is widely expressed in tissues such as thyroid and endometrium, and has been associated with Dystonia 23, making it a significant target for neurological research .

What types of PRRC1 antibodies are available for research applications?

Several types of PRRC1 antibodies are available for research purposes, primarily as rabbit polyclonal antibodies. These antibodies are typically generated using recombinant human PRRC1 protein fragments as immunogens. For example, some commercially available antibodies are produced using the Y139-D411 position fragment or the 200-445AA region of the human PRRC1 protein. Most PRRC1 antibodies are supplied in either lyophilized form or in buffer solutions containing glycerol and preservatives. They are generally unconjugated, though some may be available with fluorescent or enzymatic conjugates for specific applications .

How should researchers select the appropriate PRRC1 antibody for their specific experimental needs?

When selecting a PRRC1 antibody, researchers should consider the following methodological approach:

• Experimental application compatibility: Verify that the antibody has been validated for your specific application (WB, IF, ICC, ELISA, or flow cytometry). For instance, antibody catalog #A15083-1 is validated for Western blot at 0.1-0.25 μg/ml, Immunofluorescence at 5 μg/ml, Flow Cytometry at 1-3 μg/10^6 cells, and ELISA at 0.1-0.5 μg/ml .

• Species reactivity: Confirm that the antibody recognizes PRRC1 in your species of interest. Most available antibodies are reactive to human PRRC1, though some may cross-react with other species .

• Epitope consideration: Select antibodies targeting different epitopes if performing confirmation studies using multiple antibodies.

• Clonality: Polyclonal antibodies offer broader epitope recognition but may have batch-to-batch variability, while monoclonal antibodies provide greater consistency but may be more sensitive to epitope modifications.

• Validation data: Review the manufacturer's validation data, including Western blot images showing the expected 47 kDa band in relevant cell lines such as HeLa, MCF-7, HepG2, and Caco-2 .

What are the optimal conditions for using PRRC1 antibodies in Western blot applications?

For optimal Western blot results with PRRC1 antibodies, researchers should follow this methodological protocol:

• Sample preparation: Prepare whole cell lysates from human cell lines known to express PRRC1, such as HeLa, MCF-7, HepG2, or Caco-2. Load approximately 30 μg of protein per lane under reducing conditions .

• Gel electrophoresis: Use a 5-20% SDS-PAGE gradient gel, running at 70V for the stacking gel and 90V for the resolving gel for 2-3 hours .

• Protein transfer: Transfer proteins to a nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature to reduce non-specific binding .

• Primary antibody incubation: Dilute PRRC1 antibody to 0.1-0.25 μg/ml (or 0.5-1 μg/ml, depending on the specific antibody) in blocking solution and incubate overnight at 4°C .

• Washing: Wash the membrane with TBS-0.1% Tween three times, 5 minutes each .

• Secondary antibody: Incubate with an appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG-HRP) at a dilution of 1:5000 for 1.5 hours at room temperature .

• Detection: Develop using an enhanced chemiluminescent (ECL) detection system. The expected band for PRRC1 should appear at approximately 47 kDa .

How can immunofluorescence experiments with PRRC1 antibodies be optimized for consistent results?

To optimize immunofluorescence experiments with PRRC1 antibodies, follow these methodological guidelines:

• Cell preparation: Culture appropriate cell lines (e.g., HeLa cells) on coverslips or chamber slides until reaching 70-80% confluence .

• Fixation and permeabilization: Fix cells with 4% paraformaldehyde and permeabilize with an appropriate permeabilization buffer to facilitate antibody access to intracellular antigens .

• Antigen retrieval: Perform enzyme antigen retrieval if necessary, using an appropriate antigen retrieval reagent for 15 minutes .

• Blocking: Block with 10% goat serum (or appropriate species serum matching your secondary antibody) to minimize non-specific binding .

• Primary antibody incubation: Apply PRRC1 antibody at 5 μg/ml and incubate overnight at 4°C in a humidified chamber .

• Secondary antibody: Use a fluorochrome-conjugated secondary antibody (e.g., Cy3-conjugated goat anti-rabbit IgG) at a 1:500 dilution for 30 minutes at 37°C .

• Nuclear counterstaining: Counterstain with DAPI to visualize nuclei and provide cellular context .

• Mounting and visualization: Mount slides with anti-fade mounting medium and visualize using a fluorescence microscope with appropriate filter sets for the fluorochromes used .

• Controls: Always include primary antibody omission controls and, if possible, PRRC1 knockdown or knockout cells as negative controls.

What are the recommended protocols for using PRRC1 antibodies in flow cytometry?

For successful flow cytometry with PRRC1 antibodies, implement the following protocol:

• Cell preparation: Harvest cells of interest (e.g., U251 cells) at a concentration of approximately 1×10^6 cells per sample .

• Fixation: Fix cells with 4% paraformaldehyde to preserve cellular morphology and antigen structure .

• Permeabilization: Since PRRC1 is primarily localized in the cytoplasm, permeabilize cells with an appropriate permeabilization buffer to allow antibody access to intracellular antigens .

• Blocking: Block with 10% normal goat serum to reduce non-specific binding .

• Primary antibody: Incubate cells with PRRC1 antibody at 1-3 μg per 1×10^6 cells for 30 minutes at 20°C .

• Secondary antibody: Apply a fluorochrome-conjugated secondary antibody (e.g., DyLight®488-conjugated goat anti-rabbit IgG) at 5-10 μg per 1×10^6 cells for 30 minutes at 20°C .

• Controls: Include the following controls:

  • Isotype control: use rabbit IgG at the same concentration as the primary antibody

  • Unlabelled sample: cells with no antibody treatment

  • Single-stained controls: if performing multi-color flow cytometry

• Analysis: Analyze samples on a flow cytometer equipped with appropriate lasers and filters for the fluorochromes used. Adjust compensation settings if using multiple fluorochromes.

What are common problems encountered when using PRRC1 antibodies in Western blot, and how can they be resolved?

When troubleshooting Western blots, a systematic approach is essential. Begin by verifying the expression of PRRC1 in your sample using known positive controls. The discrepancy between the observed molecular weight (47 kDa) and calculated weight (19.9 kDa) could be due to post-translational modifications, which should be considered when analyzing results .

How can researchers validate the specificity of PRRC1 antibodies before experimental use?

To validate PRRC1 antibody specificity, researchers should implement the following comprehensive approach:

• Positive and negative control samples:

  • Use cell lines with confirmed PRRC1 expression (e.g., HeLa, MCF-7, HepG2, Caco-2) as positive controls

  • Employ PRRC1 knockdown/knockout cells (using siRNA or CRISPR) as negative controls

  • Include tissue samples known to express or lack PRRC1

• Multiple detection methods:

  • Compare results across different techniques (WB, IF, ICC, flow cytometry)

  • Verify that subcellular localization in IF/ICC matches known PRRC1 localization (cytoplasmic)

• Peptide competition assay:

  • Pre-incubate the antibody with purified PRRC1 protein or immunizing peptide

  • Verify signal reduction or elimination in Western blot or immunostaining

• Multiple antibodies comparison:

  • Compare results using antibodies targeting different PRRC1 epitopes

  • Consistent detection pattern across antibodies increases confidence in specificity

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the PRRC1 antibody and identify pulled-down proteins

  • Confirm PRRC1 presence among identified proteins

• Recombinant expression system:

  • Test antibody against cells overexpressing tagged PRRC1

  • Verify co-localization with the tag or increased signal intensity

A successful validation should demonstrate consistent detection of a 47 kDa band in Western blot analyses, appropriate subcellular localization in imaging studies, and elimination of signal in negative controls .

What is the recommended storage and handling protocol to maintain PRRC1 antibody activity?

To maintain optimal PRRC1 antibody activity, follow these storage and handling recommendations:

• Long-term storage:

  • Store lyophilized antibody at -20°C for up to one year from the date of receipt

  • For antibodies in solution, store at -20°C or -80°C, avoiding repeated freeze-thaw cycles

• Reconstitution:

  • Reconstitute lyophilized antibody by adding 0.2 ml of sterile distilled water to yield a concentration of approximately 500 μg/ml or 0.5 mg/ml

  • Gently mix by inversion; avoid vigorous shaking or vortexing that could damage the antibody

• Short-term storage after reconstitution:

  • Store reconstituted antibody at 4°C for up to one month

  • For longer storage, prepare small aliquots and store at -20°C for up to six months

• Freeze-thaw cycles:

  • Limit freeze-thaw cycles to a maximum of 3-5 times

  • Thaw aliquots completely before use and keep them on ice while working

• Working dilutions:

  • Prepare working dilutions fresh on the day of use whenever possible

  • If storage of diluted antibody is necessary, keep at 4°C for no more than 1-2 weeks

• Contamination prevention:

  • Use sterile techniques when handling antibodies

  • Some antibody solutions contain preservatives like 0.03% Proclin 300 or sodium azide to prevent microbial growth

• Transport conditions:

  • Transport on ice or with cold packs for short periods

  • For longer transportation, use dry ice to maintain frozen state

Proper storage and handling are critical for maintaining antibody functionality throughout your research project. Degraded antibodies can lead to inconsistent results and experimental failure.

How can PRRC1 antibodies be utilized to investigate PRRC1's role in Dystonia 23 pathogenesis?

To investigate PRRC1's role in Dystonia 23 pathogenesis using PRRC1 antibodies, researchers should consider these methodological approaches:

• Comparative expression analysis:

  • Use Western blot with PRRC1 antibodies to quantify PRRC1 expression levels in patient-derived samples versus controls

  • Compare PRRC1 expression in various brain regions using immunohistochemistry on post-mortem tissue

  • Implement flow cytometry with PRRC1 antibodies to assess expression in different cell populations from patient samples

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation with PRRC1 antibodies to identify interaction partners in neuronal cells

  • Use proximity ligation assays (PLA) to visualize and quantify PRRC1 interactions with other dystonia-related proteins in situ

  • Combine with mass spectrometry to identify the PRRC1 interactome in healthy versus dystonic conditions

• Subcellular localization in disease context:

  • Utilize immunofluorescence with PRRC1 antibodies to examine potential localization changes in dystonia models

  • Perform subcellular fractionation followed by Western blot to quantify PRRC1 distribution in different cellular compartments

  • Compare cytoplasmic localization patterns between healthy and affected neurons

• Disease-associated post-translational modifications:

  • Use phospho-specific or other modification-specific PRRC1 antibodies (if available) to investigate potential disease-associated modifications

  • Combine with 2D-gel electrophoresis to identify modification patterns specific to dystonia

• Functional studies:

  • Implement PRRC1 antibodies in functional blocking experiments in neuronal culture models

  • Use antibodies to monitor PRRC1 expression after genetic manipulation (overexpression, knockout, or mutation introduction)

  • Correlate PRRC1 expression with cellular phenotypes associated with dystonia

• Animal model validation:

  • Validate PRRC1 antibody reactivity in animal models of Dystonia 23

  • Use immunohistochemistry to map PRRC1 expression in brain regions affected by dystonia

  • Monitor changes in PRRC1 levels during disease progression

This multi-faceted approach would provide comprehensive insights into how PRRC1 may contribute to Dystonia 23 pathogenesis, potentially revealing new therapeutic targets.

What experimental approaches can combine PRRC1 antibodies with other molecular techniques to elucidate PRRC1 function?

Integrating PRRC1 antibodies with other molecular techniques can provide deeper insights into PRRC1 function through these advanced approaches:

• ChIP-seq (Chromatin Immunoprecipitation-sequencing):

  • If PRRC1 has nuclear functions, use ChIP-seq with PRRC1 antibodies to identify potential DNA binding sites

  • Combine with transcriptomics to correlate binding with gene expression changes

• RIME (Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins):

  • Use PRRC1 antibodies for immunoprecipitation followed by mass spectrometry

  • Identify proteins complexed with PRRC1 in different cellular contexts

  • Compare protein interaction networks between normal and disease states

• Proximity-dependent labeling combined with immunoprecipitation:

  • Express PRRC1 fused to BioID or APEX2 in cell models

  • Use PRRC1 antibodies to verify expression and localization

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

• Super-resolution microscopy:

  • Apply PRRC1 antibodies in techniques like STORM or PALM

  • Visualize nanoscale distribution and colocalization with potential interaction partners

  • Track dynamic changes in PRRC1 localization in response to stimuli

• CRISPR-Cas9 genome editing with antibody validation:

  • Generate PRRC1 knockout or mutant cell lines

  • Use PRRC1 antibodies to confirm knockout/mutation efficiency

  • Examine phenotypic consequences and rescue experiments

• Single-cell immunofluorescence combined with transcriptomics:

  • Perform immunofluorescence with PRRC1 antibodies on fixed cells

  • Correlate protein expression with single-cell RNA-seq data

  • Identify cell populations with distinctive PRRC1 expression patterns

• Tissue microarray (TMA) analysis:

  • Apply PRRC1 antibodies to TMAs containing multiple tissue types

  • Quantify expression across different tissues and disease states

  • Correlate with clinical data and patient outcomes

• FRET/FLIM (Förster Resonance Energy Transfer/Fluorescence Lifetime Imaging):

  • Label PRRC1 and potential interaction partners with suitable FRET pairs

  • Use PRRC1 antibodies to validate expression patterns

  • Measure protein-protein interactions in living cells

These integrated approaches would provide multidimensional insights into PRRC1 function beyond what could be achieved with antibody-only methods.

How do research findings on PRRC1 compare across different tissue types and experimental models?

Research findings on PRRC1 demonstrate significant variability across tissue types and experimental models, warranting careful consideration when interpreting results:

When comparing PRRC1 findings across experimental systems, researchers should consider:

• Species differences: While PRRC1 is conserved across species from mammals to zebrafish, protein function and regulation may vary. Current commercial antibodies primarily target human PRRC1 .

• Experimental technique variations: Detection sensitivity differs between Western blot (0.1-1 μg/ml), immunofluorescence (5 μg/ml), and flow cytometry (1-3 μg/10^6 cells), potentially influencing result interpretation .

• Protein characteristics: The discrepancy between calculated (19.9 kDa) and observed (47 kDa) molecular weights suggests post-translational modifications or alternative splicing that may vary across tissues .

• Disease context: PRRC1's association with Dystonia 23 indicates that its function or regulation may be altered in neurological disorders, warranting specific investigation in neuronal models .

• Subcellular distribution: While primarily cytoplasmic, PRRC1's precise distribution within cellular compartments may vary by tissue type or physiological state .

What emerging technologies might enhance PRRC1 antibody applications in research?

Several emerging technologies show promise for enhancing PRRC1 antibody applications in research:

• Single-molecule detection methods:

  • Super-resolution microscopy techniques (STORM, PALM, SIM) combined with PRRC1 antibodies could reveal nanoscale distribution patterns within the cytoplasm

  • Single-molecule pulldown (SiMPull) could enable detection of individual PRRC1 molecules and their interaction partners with unprecedented sensitivity

• Spatially-resolved proteomics:

  • CODEX (CO-Detection by indEXing) or Imaging Mass Cytometry combined with PRRC1 antibodies could map protein expression in tissues with spatial context

  • Digital Spatial Profiling (DSP) would allow quantitative measurement of PRRC1 in specific regions of tissue sections

• Engineered antibody formats:

  • Nanobodies or single-domain antibodies against PRRC1 could offer improved tissue penetration and reduced immunogenicity

  • Recombinant antibody fragments with site-specific conjugation would enable precise control over labeling stoichiometry for quantitative applications

• Mass cytometry (CyTOF):

  • Metal-labeled PRRC1 antibodies would allow multiplexed detection alongside dozens of other proteins

  • Could reveal complex relationships between PRRC1 expression and various cellular states or phenotypes

• Optogenetic applications:

  • Light-activatable antibodies against PRRC1 could enable spatiotemporal control over PRRC1 inhibition

  • Combining with live-cell imaging to observe immediate consequences of PRRC1 disruption

• Antibody-enabled proximity labeling:

  • PRRC1 antibodies conjugated to enzymes like APEX2 or TurboID could identify proximal proteins in their native cellular environment

  • Would reveal the PRRC1 microenvironment without requiring genetic modification of cells

• Microfluidic antibody-based assays:

  • Droplet-based single-cell protein analysis with PRRC1 antibodies could measure expression heterogeneity

  • Microfluidic Western blotting would enable PRRC1 analysis from limited samples like patient biopsies

• Cryo-electron tomography with immunogold labeling:

  • PRRC1 antibodies conjugated to gold nanoparticles could visualize the protein in its native cellular context at near-atomic resolution

  • Would reveal structural relationships between PRRC1 and cellular organelles

These technologies have the potential to significantly advance our understanding of PRRC1 biology by enabling more sensitive, specific, and comprehensive analyses than currently possible with standard antibody applications.

What unresolved questions about PRRC1 could be addressed using current antibody technologies?

Several critical unresolved questions about PRRC1 could be addressed using current antibody technologies:

• Functional domains and protein interactions:

  • What proteins interact with PRRC1's proline-rich N-terminus versus the DUF84 C-terminal domain?

  • This could be investigated using domain-specific antibodies in co-immunoprecipitation experiments followed by mass spectrometry

• Regulation of PRRC1 expression:

  • How does PRRC1 expression vary across tissues, developmental stages, and disease states?

  • Systematic immunohistochemistry analysis with validated PRRC1 antibodies across tissue arrays could address this question

• Post-translational modifications:

  • What modifications account for the discrepancy between predicted (19.9 kDa) and observed (47 kDa) molecular weights?

  • Immunoprecipitation with PRRC1 antibodies followed by mass spectrometry analysis could identify modifications

• Subcellular trafficking dynamics:

  • Does PRRC1 shuttle between different cellular compartments under specific conditions?

  • Live-cell imaging with fluorescently-labeled PRRC1 antibody fragments could track dynamic localization changes

• Role in Dystonia 23 pathogenesis:

  • Is PRRC1 expression, localization, or modification altered in Dystonia 23 patients?

  • Comparative immunofluorescence and Western blot analyses of patient-derived versus control samples could reveal disease-specific changes

• Functional redundancy:

  • Do other proteins compensate for PRRC1 in knockout models?

  • Antibody-based proteomics in PRRC1 knockout versus wildtype cells could identify upregulated proteins

• Cell type-specific functions:

  • Does PRRC1 serve different functions in different cell types?

  • Multiparameter flow cytometry with PRRC1 antibodies combined with cell type markers and functional readouts could address this question

• Structural organization:

  • Does PRRC1 form homo-oligomers or participate in larger protein complexes?

  • Native gel electrophoresis followed by Western blotting with PRRC1 antibodies could reveal complex formation

• Extracellular presence:

  • Is PRRC1 secreted or present in extracellular vesicles?

  • ELISA assays using PRRC1 antibodies could detect the protein in culture media or biological fluids

• Therapeutic potential:

  • Could targeting PRRC1 with therapeutic antibodies modify disease progression in Dystonia 23?

  • Functional studies using antibodies as blocking agents in cellular models could provide preliminary evidence

Addressing these questions would significantly advance our understanding of PRRC1 biology and potentially reveal new therapeutic approaches for PRRC1-associated diseases.

How might integrated multi-omics approaches incorporating PRRC1 antibody data enhance our understanding of this protein?

Integrated multi-omics approaches incorporating PRRC1 antibody data could provide comprehensive insights through the following methodological framework:

• Antibody-based proteomics integrated with transcriptomics:

  • Correlate PRRC1 protein levels (detected by antibodies in Western blot or immunohistochemistry) with mRNA expression (from RNA-seq)

  • Identify discrepancies suggesting post-transcriptional regulation

  • Example workflow: Perform Western blot quantification of PRRC1 across cell lines using antibodies at 0.1-0.25 μg/ml , then correlate with RNA-seq data from the same cells

• Spatial proteomics combined with genomics:

  • Map PRRC1 subcellular localization using immunofluorescence at 5 μg/ml antibody concentration

  • Correlate with genomic variants in PRRC1 gene (from WGS or WES)

  • Identify mutations that alter localization patterns

• Immunoprecipitation-mass spectrometry with phosphoproteomics:

  • Use PRRC1 antibodies for immunoprecipitation

  • Identify interaction partners by mass spectrometry

  • Cross-reference with phosphoproteomic data to identify phosphorylation-dependent interactions

  • Create interaction networks revealing PRRC1's role in signaling pathways

• Antibody-based flow cytometry with single-cell transcriptomics:

  • Sort cells based on PRRC1 expression levels using flow cytometry with PRRC1 antibodies at 1-3 μg/10^6 cells

  • Perform single-cell RNA-seq on sorted populations

  • Identify gene expression signatures associated with different PRRC1 expression levels

• Chromatin immunoprecipitation with metabolomics:

  • If PRRC1 has nuclear functions, perform ChIP-seq with PRRC1 antibodies

  • Correlate binding sites with metabolomic changes in PRRC1-depleted cells

  • Identify metabolic pathways potentially regulated by PRRC1

• Multi-parameter imaging with lipidomics:

  • Perform multicolor immunofluorescence with PRRC1 antibodies and organelle markers

  • Correlate PRRC1 localization with lipidomic profiles

  • Investigate potential roles in lipid metabolism or membrane organization

• Antibody-based quantitative proteomics with structural biology:

  • Use PRRC1 antibodies to quantify expression in native versus stress conditions

  • Combine with structural predictions or experimental structures of PRRC1

  • Identify structure-function relationships in different cellular states

• Systems biology integration:

  • Compile all antibody-derived PRRC1 data (expression, localization, interactions)

  • Integrate with multi-omics datasets in a systems biology model

  • Create a comprehensive PRRC1 functional network

A sample data integration table might look like:

This multi-omics integration would provide unprecedented insights into PRRC1 function, potentially revealing its role in health and disease contexts beyond what any single approach could achieve.