LRPPRC Antibody, FITC conjugated

What is LRPPRC and what cellular functions does it perform?

LRPPRC (Leucine-rich PPR-motif containing protein, also known as LRP130 or GP130) is a 130-kDa RNA-binding protein belonging to the pentatricopeptide repeat family. This multifunctional protein primarily localizes to the mitochondria where it binds to poly(A) mRNA and plays a crucial role in the translation or stability of mitochondrially encoded cytochrome c oxidase (COX) subunits . Beyond its mitochondrial functions, LRPPRC also regulates nuclear gene transcription and binds specific RNA molecules in both the nucleus and cytoplasm . The protein has a calculated molecular weight of 158 kDa (1394 amino acids) but typically appears at approximately 130 kDa in experimental analyses . Mutations in the LRPPRC gene are notably associated with the French-Canadian type of Leigh syndrome, underscoring its physiological importance .

What are the advantages of using FITC-conjugated LRPPRC antibodies?

FITC-conjugated LRPPRC antibodies offer several methodological advantages for research applications:

• Direct visualization without secondary antibodies, reducing experimental steps and potential cross-reactivity issues

• Compatibility with live-cell imaging applications (with appropriate protocols)

• Well-established excitation/emission profile (approximately 493 nm / 522 nm), compatible with standard FITC filter sets in microscopy and flow cytometry instrumentation

• Ability to perform multiplexing with antibodies conjugated to spectrally distinct fluorophores

• Reduced background compared to indirect detection methods when optimized properly

The direct conjugation to fluorescent dyes enables straightforward detection in applications such as immunofluorescence, immunocytochemistry, and flow cytometry, streamlining experimental workflows while maintaining specificity for the target protein .

What dilution ranges are recommended for FITC-conjugated LRPPRC antibodies in different applications?

Optimal dilution ranges for FITC-conjugated LRPPRC antibodies vary by application and specific antibody product. The following table summarizes recommended dilutions based on extensive validation:

It is strongly recommended that researchers titrate the antibody in each testing system to obtain optimal results, as sensitivity can vary significantly based on sample type, fixation method, and detection instrumentation . As a general approach, start with mid-range dilutions and adjust based on signal-to-noise ratio in preliminary experiments.

What are the optimal storage conditions for maintaining FITC-conjugated LRPPRC antibodies?

For maximum stability and performance of FITC-conjugated LRPPRC antibodies, adhere to these storage guidelines:

• Store at -20°C in the buffer provided (typically PBS with 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3)

• Consistently avoid exposure to light to prevent photobleaching of the FITC fluorophore

• Most commercially available preparations remain stable for one year after shipment when properly stored

• Contrary to some standard antibody protocols, aliquoting is generally unnecessary for -20°C storage of these preparations based on manufacturer recommendations

Improper storage, particularly exposure to light or temperature fluctuations, can lead to reduced fluorescence intensity and compromised experimental results. After thawing for use, maintain the antibody on ice and protected from light throughout the experimental procedure.

How should researchers design appropriate controls for experiments with FITC-conjugated LRPPRC antibodies?

Rigorous control design is essential for accurate interpretation of results with FITC-conjugated LRPPRC antibodies:

• Isotype Control: Include an irrelevant FITC-conjugated antibody of the same isotype (e.g., Rabbit IgG or Mouse IgG2b depending on the specific LRPPRC antibody) to assess non-specific binding.

• Negative Controls:

  • Unstained samples to establish autofluorescence baseline

  • Secondary-only controls (for comparative experiments using unconjugated primary antibodies)

  • Cells known to express minimal LRPPRC (if available)

• Positive Controls:

  • HEK-293 or HeLa cells, which have been validated to express detectable levels of LRPPRC

• Validation Controls:

  • Competitive blocking with the immunogen peptide

  • Parallel staining with alternative LRPPRC antibody clones or unconjugated versions

  • RNA interference to demonstrate specificity via reduced signal after LRPPRC knockdown

These controls collectively ensure that observed fluorescence signals genuinely represent LRPPRC distribution rather than artifacts or non-specific binding.

What is the recommended protocol for immunofluorescence using FITC-conjugated LRPPRC antibodies?

The following optimized protocol is recommended for immunofluorescence applications with FITC-conjugated LRPPRC antibodies:

• Cell Preparation and Fixation:

  • Culture cells on appropriate coverslips or chamber slides

  • Wash cells twice with PBS (pH 7.4)

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash three times with PBS (5 minutes each)

• Permeabilization and Blocking:

  • Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

  • Wash three times with PBS (5 minutes each)

  • Block with 5% normal serum (from the same species as the secondary antibody) in PBS for 1 hour at room temperature

• Antibody Incubation:

  • Dilute FITC-conjugated LRPPRC antibody in antibody dilution buffer (1% BSA in PBS) at 1:50-1:500 dilution

  • Incubate cells with diluted antibody overnight at 4°C in a humidified chamber protected from light

  • Wash five times with PBS (5 minutes each)

• Counterstaining and Mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

  • Wash three times with PBS (5 minutes each)

  • Mount slides with anti-fade mounting medium

  • Seal edges with nail polish and store at 4°C protected from light

• Imaging:

  • Use appropriate filter sets for FITC (excitation ~493 nm, emission ~522 nm)

  • Capture images with consistent exposure settings across samples

This protocol can be modified based on specific experimental requirements and cell types. For co-localization studies, additional primary antibodies with compatible fluorophores can be included in the primary antibody incubation step.

How can researchers optimize FITC-conjugated LRPPRC antibody protocols for flow cytometry?

For optimal flow cytometry results with FITC-conjugated LRPPRC antibodies, implement the following specialized protocol:

• Cell Preparation:

  • Harvest cells using appropriate methods (trypsinization or scraping)

  • Wash twice with ice-cold PBS

  • Count cells and aliquot 1×10^6 cells per sample

• Fixation and Permeabilization (for intracellular staining):

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash twice with PBS

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 in PBS for 15 minutes at room temperature

  • Wash twice with PBS

• Blocking and Staining:

  • Block with 5% normal serum in PBS for 30 minutes at room temperature

  • Add FITC-conjugated LRPPRC antibody at 0.80 μg per 10^6 cells in 100 μl suspension

  • Incubate for 30-60 minutes at room temperature in the dark

  • Wash three times with PBS

• Analysis:

  • Resuspend cells in 400-500 μl PBS

  • Analyze using flow cytometer with appropriate settings for FITC detection

  • Include appropriate controls as described in section 2.3

For optimal signal-to-noise ratio, researchers should carefully titrate the antibody concentration and adjust permeabilization conditions based on preliminary experiments. Remember that overly harsh permeabilization may disrupt mitochondrial structures and affect LRPPRC localization patterns.

What approaches can resolve common troubleshooting issues with FITC-conjugated LRPPRC antibodies?

When encountering challenges with FITC-conjugated LRPPRC antibodies, consider these methodological solutions:

For persistent issues, consider comparing results with unconjugated LRPPRC antibodies followed by secondary detection to determine if the problem lies with the FITC conjugation or with the primary antibody specificity itself .

How can FITC-conjugated LRPPRC antibodies be used for co-localization studies with other mitochondrial markers?

FITC-conjugated LRPPRC antibodies can be effectively integrated into multi-color imaging experiments to investigate mitochondrial biology:

• Compatible Mitochondrial Markers:

  • MitoTracker dyes (Red or Deep Red variants to avoid spectral overlap)

  • Antibodies against TOMM20, COX IV, or cytochrome c (conjugated to spectrally distinct fluorophores like Cy3, Cy5, or Alexa Fluor 647)

  • Genetically encoded markers (e.g., mito-DsRed or mito-mCherry) in transfected cells

• Experimental Design Considerations:

  • Select fluorophores with minimal spectral overlap for clear channel separation

  • Perform single-color controls to establish bleed-through parameters

  • Consider sequential rather than simultaneous acquisition if spectral overlap is a concern

  • Use appropriate excitation/emission filters optimized for FITC (493 nm / 522 nm)

• Quantitative Co-localization Analysis:

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Perform intensity correlation analysis

  • Use specialized software (ImageJ with Coloc2 plugin, Imaris, or similar tools)

By combining FITC-conjugated LRPPRC antibodies with other mitochondrial markers, researchers can investigate LRPPRC's precise suborganellar localization and its potential interactions with other mitochondrial components or compartments.

What considerations are important when studying LRPPRC in disease models?

When investigating LRPPRC in disease models, particularly those related to mitochondrial dysfunction, consider these methodological approaches:

• French-Canadian Leigh Syndrome Studies:

  • Compare LRPPRC expression levels and localization patterns between patient-derived and control cells

  • Correlate LRPPRC levels with COX activity and mitochondrial mRNA stability

  • Investigate potential changes in LRPPRC post-translational modifications

• Other Mitochondrial Diseases:

  • Examine LRPPRC expression in models of OXPHOS deficiency

  • Investigate relationships between LRPPRC levels and mitochondrial translation

  • Study LRPPRC interaction with other mitochondrial RNA-binding proteins

• Cancer Research Applications:

  • Evaluate LRPPRC expression across cancer cell lines and tumor samples

  • Assess correlation between LRPPRC levels and metabolic phenotypes

  • Investigate LRPPRC's role in mitochondrial adaptations during carcinogenesis

• Neurodegenerative Disorders:

  • Examine LRPPRC expression in models of Parkinson's, Alzheimer's, or ALS

  • Study relationships between LRPPRC function and mitochondrial dynamics

For disease-related research, combining FITC-conjugated LRPPRC antibody staining with functional readouts of mitochondrial function (membrane potential, ROS production, ATP synthesis) can provide mechanistic insights into LRPPRC's role in pathophysiology.

How can FITC-conjugated LRPPRC antibodies be validated for specificity in experimental systems?

Comprehensive validation of FITC-conjugated LRPPRC antibodies should incorporate multiple complementary approaches:

• Genetic Validation:

  • LRPPRC knockout or knockdown cells as negative controls

  • LRPPRC overexpression systems as positive controls

  • Rescue experiments reintroducing LRPPRC in knockout backgrounds

• Biochemical Validation:

  • Western blot analysis with the unconjugated version of the same antibody

  • Immunoprecipitation followed by mass spectrometry

  • Peptide competition assays with the immunizing peptide

• Cross-Validation:

  • Compare staining patterns using multiple LRPPRC antibodies targeting different epitopes

  • Correlation with mRNA expression data from RT-PCR or RNA-seq

  • Comparison with GFP-tagged LRPPRC expression in transfected cells

• Expected Molecular Weight Confirmation:

  • Verify detection at the expected molecular weight (observed ~130 kDa vs. calculated 158 kDa)

  • Ensure absence of non-specific bands in western blot validations

Thorough validation is particularly important when applying these antibodies to new model systems or species beyond the explicitly tested human, mouse, and rat samples .

What are the key technical specifications of available FITC-conjugated LRPPRC antibodies?

The following table summarizes the technical specifications of commercially available FITC-conjugated LRPPRC antibodies:

*Note: CL594-67679 is not FITC-conjugated but is included for comparative purposes as it represents an alternative fluorophore option for LRPPRC detection .

These specifications are crucial for determining compatibility with experimental systems and imaging equipment. Researchers should consider these parameters when selecting the most appropriate antibody for their specific applications.

How does LRPPRC protein structure influence epitope availability and antibody selection?

LRPPRC protein structure has significant implications for antibody epitope accessibility and experimental design:

• Domain Organization:

  • LRPPRC contains numerous pentatricopeptide repeat (PPR) motifs that form alpha-helical structures

  • The protein has a calculated molecular weight of 158 kDa (1394 amino acids)

  • Different antibodies target distinct regions, with ABIN7158047 specifically targeting amino acids 901-1036

• Epitope Considerations:

  • Epitope accessibility may vary depending on LRPPRC's conformational state

  • Protein-protein interactions may mask certain epitopes

  • Fixation methods can differentially affect epitope exposure

• Selection Guidance:

  • For studying full-length LRPPRC, antibodies recognizing conserved regions are preferable

  • For isoform-specific detection, select antibodies targeting unique regions

  • Consider using antibodies validated in your specific application (IF/ICC, FC)

• Cross-Reactivity:

  • Due to sequence conservation, some antibodies show reactivity across human, mouse, and rat samples

  • Species-specific validation is recommended when working with models not explicitly tested

Understanding these structural considerations helps researchers select the most appropriate FITC-conjugated LRPPRC antibody for their specific experimental questions and systems.

What are emerging applications for FITC-conjugated LRPPRC antibodies in cutting-edge research?

FITC-conjugated LRPPRC antibodies are increasingly being applied in innovative research directions:

• Single-Cell Analysis:

  • Integration with single-cell RNA-seq to correlate protein localization with transcriptional profiles

  • Application in microfluidic systems for high-throughput screening

  • Combination with super-resolution microscopy techniques for nanoscale localization analysis

• Live-Cell Imaging Adaptations:

  • Development of cell-permeable nanobody-based detection systems

  • Investigation of LRPPRC dynamics during mitochondrial stress responses

  • Real-time tracking of LRPPRC redistribution during cellular perturbations

• Multi-Omics Integration:

  • Correlation of LRPPRC localization with proteomics and metabolomics data

  • Spatial transcriptomics approaches to map LRPPRC-associated RNA species

  • Systems biology modeling of LRPPRC's role in mitochondrial homeostasis

• Therapeutic Development:

  • Screening for compounds that modulate LRPPRC function or localization

  • Exploration of LRPPRC as a potential biomarker in mitochondrial diseases

  • Investigation of LRPPRC-targeting approaches for diseases with mitochondrial dysfunction

These emerging applications highlight the continued relevance of FITC-conjugated LRPPRC antibodies in advancing our understanding of mitochondrial biology and associated pathologies.

What methodological advances might improve FITC-conjugated LRPPRC antibody applications?

Future methodological improvements may enhance the utility of FITC-conjugated LRPPRC antibodies:

• Technical Enhancements:

  • Development of brighter and more photostable FITC derivatives

  • Creation of pH-insensitive variants to improve performance in acidic cellular compartments

  • Generation of switchable fluorophores for super-resolution applications

• Application Expansions:

  • Optimization for tissue clearing techniques and 3D imaging

  • Adaptation for multiplexed tissue imaging platforms

  • Integration with automated high-content screening systems

• Validation Approaches:

  • Establishment of standardized validation protocols across research communities

  • Creation of knockout cell line panels for definitive specificity testing

  • Development of synthetic biology standards for absolute quantification