LRPPRC Antibody

What applications can LRPPRC antibodies be used for in research?

LRPPRC antibodies have been validated for multiple research applications, with specific dilution recommendations for optimal results:

• Western Blot (WB): 1:5000-1:50000 dilution

• Immunohistochemistry (IHC): 1:50-1:500 dilution

• Immunofluorescence (IF/ICC): 1:200-1:800 dilution

• Immunoprecipitation (IP): 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Co-Immunoprecipitation (CoIP)

• RNA Immunoprecipitation (RIP)

• ELISA

These applications enable researchers to detect LRPPRC protein expression, localization, and interactions with other proteins or RNA molecules.

What species reactivity do commercial LRPPRC antibodies demonstrate?

Most commercial LRPPRC antibodies show reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species. Some antibodies have been further validated to react with samples from additional species:

When planning cross-species experiments, researchers should verify the antibody's reactivity with their specific samples through preliminary validation tests .

How should I prepare samples for LRPPRC protein detection via Western blot?

For optimal Western blot detection of LRPPRC:

• Prepare cell/tissue lysates using a standard lysis buffer (e.g., 150 mM NaCl, 1.0% Nonidet P40, 50 mM Tris/HCl, pH 8.0)

• Quantify protein concentration using a BCA protein assay kit

• Load equal amounts of protein (typically 10-30 μg per lane)

• Separate proteins on 10% SDS-PAGE (LRPPRC is a large protein with observed molecular weight of ~130 kDa)

• Transfer to PVDF or nitrocellulose membrane

• Block with 5% nonfat milk in TBST buffer

• Incubate with LRPPRC primary antibody at recommended dilutions (1:5000-1:50000) overnight at 4°C

• Wash with TBST buffer 3-5 times

• Incubate with HRP-conjugated secondary antibody

• Develop using ECL detection reagents

This protocol has been successfully used to detect LRPPRC in various cell lines including HeLa, HEK-293T, HepG2, and tissue samples from different organs.

How can I effectively use LRPPRC antibodies for co-immunoprecipitation studies?

For co-immunoprecipitation studies investigating LRPPRC interactions with partner proteins:

• Prepare cell lysates from 100-mm dishes using a gentle lysis buffer (150 mM NaCl, 1.0% Nonidet P40, 50 mM Tris/HCl, pH 8.0)

• Quantify total protein concentration

• Use 1.6 mg of total protein for each immunoprecipitation reaction

• Add 2 μg of LRPPRC antibody (or antibodies against potential interacting partners like Bcl-2 or Beclin 1)

• Include appropriate IgG control antibodies for specificity validation

• Incubate with Protein G-agarose beads

• Wash precipitates extensively (5 times) with lysis buffer

• Resuspend precipitates in lysis buffer containing loading buffer

• Boil for 5 minutes before SDS-PAGE separation

• Perform immunoblotting for potential interacting proteins

This approach has successfully demonstrated interactions between LRPPRC and proteins involved in autophagy regulation, such as the LRPPRC-Beclin 1-Bcl-2 ternary complex .

What are the key considerations when designing LRPPRC knockdown experiments?

When designing LRPPRC knockdown experiments to study its function:

• Choose appropriate knockdown method:

  • siRNA for transient knockdown effects

  • shRNA for stable knockdown via lentiviral delivery

  • CRISPR-Cas9 for complete knockout

• Consider validated sequences:

  • For shLRPPRC: hU6-MCS-CBh-gcGFP-IRES-puromycin vector system has been validated

  • For siRNA: sequences derived from validated shRNA can be synthesized

• Include proper controls:

  • Empty vector controls for overexpression studies

  • Non-targeting siRNA/shRNA controls for knockdown studies

• Verify knockdown efficiency:

  • Western blot using validated LRPPRC antibodies

  • qRT-PCR for mRNA level assessment

• Assess phenotypic changes relevant to LRPPRC function:

  • Mitochondrial function (membrane potential, respiratory capacity)

  • Expression of mitochondrial DNA-encoded genes

  • Autophagy markers (LC3-I/II, p62)

  • Glycolytic parameters in cancer studies

• Consider rescue experiments:

  • Re-express wildtype or mutant LRPPRC to confirm specificity of phenotypes

Researchers should note that complete LRPPRC knockout can be embryonically lethal in mice, so tissue-specific or inducible systems may be preferable for in vivo studies .

How can LRPPRC antibodies be used to study mitochondrial disease mechanisms?

LRPPRC antibodies are valuable tools for investigating mitochondrial disease mechanisms, particularly in Leigh syndrome, French-Canadian type (LSFC):

• Comparative expression analysis:

  • Compare LRPPRC protein levels in patient-derived vs. control cells using Western blot

  • Assess subcellular localization changes via immunofluorescence

• Functional impact assessment:

  • Immunoprecipitate LRPPRC to identify differential protein interactions in disease models

  • Use LRPPRC antibodies alongside mitochondrial markers to evaluate morphological changes

• Therapeutic target validation:

  • Monitor LRPPRC levels during drug treatments aimed at improving mitochondrial function

  • Assess restoration of LRPPRC-dependent pathways following interventions

• Disease model validation:

  • Verify LRPPRC expression in LSFC patient-derived cells or tissues

  • Validate CRISPR-engineered disease models carrying the 1119C>T LSFC mutation

• Study mitoribosomal interactions:

  • Investigate associations between LRPPRC and mitoribosomal proteins mS39 and mS31

  • Examine the impact of disease mutations on these interactions

LRPPRC antibodies can help determine if pathogenic mechanisms involve altered protein expression, localization, or interactions with binding partners.

What is the role of LRPPRC in cancer, and how can antibodies help investigate this function?

LRPPRC has emerging roles in cancer progression, and antibodies provide crucial tools for investigating these functions:

• Expression analysis in tumors:

  • Evaluate LRPPRC protein levels in tumor vs. normal tissues via IHC and Western blot

  • Correlate expression with clinical parameters and patient outcomes

• Metabolic reprogramming studies:

  • Use LRPPRC antibodies to study its involvement in glycolysis regulation

  • Investigate interactions with metabolic enzymes like LDHA

• Immune evasion mechanisms:

  • Explore correlations between LRPPRC and PD-L1 expression

  • Assess impact on tumor-infiltrating lymphocytes (CD8+, CD4+ T cells)

  • Examine relationships with chemokines CXCL9 and CXCL10

• mRNA modification analysis:

  • Investigate LRPPRC's role as an m6A modification reader

  • Study its impact on the stability of cancer-related mRNAs

• Signaling pathway investigation:

  • Examine LRPPRC's roles in PI3K/Akt/mTOR signaling

  • Study interactions with autophagy regulators Beclin 1 and Bcl-2

For cancer xenograft models, LRPPRC antibodies have been successfully used for analyzing protein expression and correlating it with immune infiltration markers via immunohistochemistry .

What are common troubleshooting strategies for Western blot detection of LRPPRC?

When troubleshooting Western blot detection of LRPPRC:

• No signal or weak signal:

  • Increase antibody concentration (start with 1:5000 and adjust as needed)

  • Extend primary antibody incubation time (overnight at 4°C recommended)

  • Increase protein loading (30-50 μg for low-expressing samples)

  • Use more sensitive ECL detection systems

  • Verify sample preparation (avoid excessive heating which may degrade large proteins)

• Multiple bands:

  • Optimize blocking conditions (5% nonfat milk in TBST recommended)

  • Increase washing steps (5× with TBST)

  • Use freshly prepared samples to avoid degradation

  • Verify antibody specificity with knockout/knockdown controls

• High background:

  • Increase dilution of primary antibody (up to 1:50000 for high-expressing samples)

  • Extend washing steps (5× with TBST buffer)

  • Optimize secondary antibody dilution (1:5000-1:10000)

• Unexpected molecular weight:

  • Note that while calculated molecular weight of LRPPRC is 158 kDa, it typically runs at ~130 kDa on SDS-PAGE

  • Check for tissue-specific or disease-specific variants

For validation, LRPPRC antibodies have been confirmed to detect the protein in various cell lines including HCT 116, HepG2, HeLa, and SH-SY5Y cells .

How can I validate the specificity of an LRPPRC antibody for my research?

To validate LRPPRC antibody specificity for your specific research application:

• Positive and negative controls:

  • Use cell lines known to express LRPPRC (HeLa, HepG2, HEK-293T cells)

  • Include tissues with known high (heart, skeletal muscle, kidney, liver) and low expression (lung, small intestine)

  • Generate LRPPRC knockdown/knockout samples as negative controls

• Antibody validation techniques:

  • Perform Western blot to confirm single band at expected molecular weight (~130 kDa)

  • Conduct peptide competition assay with immunizing peptide

  • Compare results from multiple LRPPRC antibodies targeting different epitopes

  • Verify subcellular localization patterns (mitochondrial) via immunofluorescence

• Application-specific validation:

  • For IHC: include appropriate positive tissue controls (heart, kidney, liver)

  • For IF/ICC: confirm co-localization with mitochondrial markers

  • For IP: verify identity of precipitated protein by mass spectrometry

• Method-specific considerations:

  • For IF/ICC: test different fixation methods (paraformaldehyde recommended)

  • For IHC: optimize antigen retrieval (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • For IP: determine optimal antibody-to-lysate ratio

Publications using knockout/knockdown controls provide strong validation for antibody specificity, with several studies using LRPPRC-KO cell lines to confirm antibody performance .

How can LRPPRC antibodies be used to investigate mitochondrial RNA metabolism?

LRPPRC antibodies are valuable tools for studying mitochondrial RNA metabolism:

• RNA immunoprecipitation (RIP) assays:

  • Immunoprecipitate LRPPRC-RNA complexes using validated antibodies

  • Identify bound RNA species via RT-qPCR or sequencing

  • Analyze changes in RNA binding under different conditions

• Post-transcriptional regulation studies:

  • Examine LRPPRC's role in stabilizing mitochondrial mRNAs

  • Investigate polyadenylation of mitochondrial transcripts

  • Study the LRPPRC-SLIRP complex and its RNA-binding properties

• Mitochondrial translation regulation:

  • Analyze LRPPRC's interaction with mitoribosomal proteins

  • Investigate how LRPPRC affects translation patterns of mitochondrial mRNAs

  • Study the LRPPRC-dependent coordination between mitochondrial transcription and translation

• Disease-relevant RNA metabolism:

  • Examine how pathogenic mutations affect LRPPRC's RNA-binding properties

  • Investigate mitochondrial mRNA stability in disease models

  • Study the impact of LRPPRC deficiency on specific mitochondrial transcripts

Research has shown that LRPPRC specifically affects mitochondrial DNA-encoded mRNAs but not rRNAs, suggesting a selective role in post-transcriptional regulation of certain mitochondrial transcripts .

What methodological approaches can be used to study LRPPRC's role in autophagy regulation?

To investigate LRPPRC's role in autophagy regulation:

• Protein interaction studies:

  • Use co-immunoprecipitation with LRPPRC antibodies to analyze interactions with autophagy-related proteins (Beclin 1, Bcl-2)

  • Perform proximity ligation assays to visualize protein interactions in situ

  • Investigate the formation of the LRPPRC-Beclin 1-Bcl-2 ternary complex

• Autophagy flux assessment:

  • Monitor LC3-I to LC3-II conversion via Western blot following LRPPRC manipulation

  • Analyze p62 levels as markers of autophagy degradation

  • Use GFP-LC3 puncta formation assays to visualize autophagosome formation

• Mitochondrial quality control:

  • Assess mitochondrial membrane potential using fluorescent dyes

  • Monitor mitophagy markers following LRPPRC knockdown/overexpression

  • Analyze mitochondrial morphology changes via electron microscopy

• Signaling pathway analysis:

  • Study the effects of LRPPRC on the PI3K/Akt/mTOR pathway

  • Examine how LRPPRC affects the ATG5-ATG12 conjugation system

  • Investigate whether LRPPRC's role in autophagy is upstream or downstream of specific autophagy regulators

Research has demonstrated that LRPPRC suppression leads to reduced mitochondrial potential, decreased Bcl-2 levels, and enhanced autophagy activation, suggesting LRPPRC functions as a negative regulator of basal autophagy .

How can I design experiments to investigate LRPPRC's role in immune regulation and cancer progression?

To investigate LRPPRC's role in immune regulation and cancer progression:

• Expression analysis in tumor microenvironment:

  • Perform multiplex immunohistochemistry for LRPPRC alongside immune markers

  • Use tissue microarrays to analyze LRPPRC expression across tumor samples

  • Correlate LRPPRC levels with patient outcomes and treatment responses

• Immune checkpoint regulation:

  • Investigate LRPPRC's relationship with PD-L1 expression via co-expression analysis

  • Study how LRPPRC knockdown affects PD-L1 expression and immune cell infiltration

  • Examine LRPPRC's role in m6A modification of immune-related mRNAs

• T-cell infiltration and activation studies:

  • Compare CD8+ and CD4+ T-cell densities in tumors with high vs. low LRPPRC expression

  • Analyze chemokine expression (CXCL9, CXCL10) in relation to LRPPRC levels

  • Study T-cell activation markers in co-culture systems with LRPPRC-manipulated cancer cells

• In vivo models:

  • Generate LRPPRC knockout xenograft models

  • Analyze tumor growth, immune infiltration, and treatment responses

  • Perform adoptive T-cell transfer experiments to evaluate immune rejection

• Combination therapy assessment:

  • Test combinations of LRPPRC targeting with immune checkpoint inhibitors

  • Evaluate synergistic effects on tumor growth and immune infiltration

  • Study mechanisms of potential synthetic lethality

Research has shown that LRPPRC deficiency enhances anti-tumor immunity, with LRPPRC knockout tumors showing increased CD8+ and CD4+ T-cell infiltration and altered chemokine expression .

What is the relationship between LRPPRC and m6A RNA modification, and how can antibodies help study this function?

The relationship between LRPPRC and m6A RNA modification is an emerging area of research:

• m6A reader function assessment:

  • Use LRPPRC antibodies in conjunction with m6A antibodies for co-localization studies

  • Perform RNA immunoprecipitation with LRPPRC antibodies followed by m6A analysis

  • Investigate LRPPRC binding to m6A-modified transcripts

• Target identification approaches:

  • Conduct MeRIP-sequencing after LRPPRC immunoprecipitation

  • Compare m6A profiles in LRPPRC-depleted vs. control cells

  • Identify specific mRNAs regulated by LRPPRC in an m6A-dependent manner

• RNA stability assessment:

  • Measure half-lives of m6A-modified transcripts in LRPPRC-manipulated cells

  • Study how LRPPRC affects the stability of specific mRNAs like LDHA

  • Investigate mechanisms of post-transcriptional regulation

• Protein interaction network:

  • Identify interactions between LRPPRC and other m6A regulatory proteins

  • Study potential cooperative or competitive relationships with other m6A readers

  • Examine how these interactions affect target mRNA fate

Immunofluorescence studies have demonstrated that LRPPRC can co-localize with m6A, supporting its role as an m6A reader protein. Research has shown that LRPPRC promotes glycolysis by stabilizing LDHA mRNA via m6A modification, contributing to metabolic reprogramming in cancer .

How can I design experiments to investigate LRPPRC's role in mitochondrial translation using antibodies?

To investigate LRPPRC's role in mitochondrial translation:

• Mitoribosome interaction studies:

  • Use LRPPRC antibodies for immunoprecipitation followed by mass spectrometry

  • Investigate interactions with mitoribosomal proteins mS39 and mS31

  • Perform proximity ligation assays to visualize these interactions in situ

• Translation pattern analysis:

  • Study mitochondrial translation products via metabolic labeling in LRPPRC-manipulated cells

  • Analyze changes in specific mitochondrially-encoded proteins via Western blot

  • Examine translation efficiency of individual mitochondrial mRNAs

• LRPPRC-SLIRP complex investigations:

  • Use antibodies against both LRPPRC and SLIRP to study their cooperative functions

  • Analyze how disruption of this complex affects mitochondrial translation

  • Investigate structural aspects of the LRPPRC-SLIRP interaction

• Disease-relevant translation studies:

  • Compare translation patterns in cells expressing wildtype vs. mutant LRPPRC

  • Study how LSFC mutations affect LRPPRC's association with the mitoribosome

  • Investigate translation defects in patient-derived samples

Recent research has revealed that LRPPRC associates with mitoribosomal proteins through recognition of LRPPRC helical repeats, suggesting a direct role in translation regulation beyond its RNA-binding functions .

What quantitative methods can be used with LRPPRC antibodies for comparative expression analysis?

For quantitative analysis of LRPPRC expression across different samples:

• Western blot densitometry:

  • Use LRPPRC antibodies at consistent dilutions (1:5000-1:10000 recommended)

  • Include loading controls (β-actin) for normalization

  • Measure band intensity using software like ImageJ

  • Calculate relative expression levels compared to controls

• Quantitative immunohistochemistry:

  • Use automated staining platforms for consistency

  • Apply recommended dilutions (1:50-1:500)

  • Employ digital pathology software for quantification

  • Score staining intensity and percentage of positive cells

• Immunofluorescence quantification:

  • Use consistent antibody dilutions (1:200-1:800)

  • Capture images with identical exposure settings

  • Measure fluorescence intensity in defined cellular regions

  • Quantify co-localization with mitochondrial markers

• High-throughput analysis:

  • Develop ELISA-based quantification methods

  • Utilize cytometric bead array technologies

  • Create tissue microarrays for parallel sample analysis

  • Apply machine learning algorithms for pattern recognition

Quantitative approaches have been successfully used to correlate LRPPRC expression with various clinical parameters, including tumor progression and patient survival. The Pearson correlation coefficient between qRT-PCR and microarray analysis for LRPPRC has been reported as 0.99, indicating high reliability of quantitative measurements .

What are the best practices for designing and analyzing LRPPRC knockout models?

When designing and analyzing LRPPRC knockout models:

• Knockout strategy selection:

  • Consider complete KO may be embryonically lethal (use tissue-specific or inducible systems)

  • Target specific functional domains to study partial loss-of-function

  • Create allelic series with different knockdown efficiencies for dose-dependent studies

• Validation approaches:

  • Confirm knockout at genomic level (sequencing)

  • Verify protein loss using validated LRPPRC antibodies via Western blot

  • Check mRNA levels using qRT-PCR

  • Include multiple control cell lines

• Phenotypic characterization:

  • Analyze mitochondrial gene expression via RNA-seq

  • Assess respiratory chain function with biochemical assays

  • Examine mitochondrial morphology via microscopy

  • Study cell growth, metabolism, and stress responses

• Data interpretation considerations:

  • Use gene set enrichment analysis to identify affected pathways

  • Focus on both direct and indirect effects of LRPPRC loss

  • Consider tissue-specific or cell type-specific responses

  • Correlate findings with patient data when available

• Rescue experiments:

  • Re-express wildtype LRPPRC to confirm phenotype specificity

  • Introduce LSFC mutation (1119C>T) to study pathogenic mechanisms

  • Test domain-specific mutants to map functional regions

Studies have successfully used GSEA on expression data from engineered LSFC cell lines with progressively decreased LRPPRC expression, identifying seven gene sets significantly correlated with LRPPRC expression .