GRPEL1 Antibody

What is GRPEL1 and what is its biological significance?

GRPEL1 (GrpE Like 1, Mitochondrial) is a protein coding gene that functions as an essential nucleotide exchange factor (NEF) in mammalian mitochondria. It serves as a cochaperone of mitochondrial Hsp70 (mtHsp70) and plays a crucial role in mitochondrial protein import and folding processes .

GRPEL1 is involved in several key cellular pathways:

• Metabolism of proteins

• Mitochondrial protein import

• Protein folding in cooperation with LONP1 and mtHSP70

Notably, GRPEL1 cannot be compensated by its paralog GRPEL2, making it essential for mitochondrial function . Diseases associated with GRPEL1 include Human Monocytic Ehrlichiosis and Lymphangitis .

How are GRPEL1 antibodies typically generated and what are their key specifications?

GRPEL1 antibodies are typically generated using specific immunogens that represent regions of the human GRPEL1 protein. Based on the available data, most commercial GRPEL1 antibodies are:

• Host/Isotype: Predominantly rabbit IgG polyclonal antibodies

• Immunogen Sequences: Often targeting specific peptide sequences such as "VLEKATQCVPKEEIKDDNPHLKNLYEGLVMTEVQIQKVFTKHGLLKLNPVGAKFDPYEHEALFHTPVEGKEPGTVALVSKVGYK"

• Purification Method: Typically affinity purified

• Storage Buffer: Usually in PBS with preservatives such as sodium azide and glycerol

• Reactivity: Most show cross-reactivity with human, mouse, and rat samples

The calculated molecular weight of human GRPEL1 is approximately 24 kDa (217 amino acids) , which is important for validating antibody specificity.

What are the principal applications for GRPEL1 antibodies in research?

GRPEL1 antibodies have been validated for multiple experimental applications:

These applications enable researchers to investigate GRPEL1 expression, localization, and interactions in various experimental contexts .

How should I design experiments to investigate GRPEL1 interactions with other mitochondrial proteins?

When investigating GRPEL1 interactions with other mitochondrial proteins, consider the following methodological approaches:

• Co-immunoprecipitation (Co-IP): Use Pierce Crosslink Immunoprecipitation Kit or similar, followed by LC-MS/MS analysis to identify interacting partners . This approach has successfully identified interactions between GRPEL1 and other proteins in previous studies.

• GST Pull-down Assays: Generate GST-tagged GRPEL1 fusion proteins (or domains) to investigate direct protein-protein interactions. Express these in E. coli systems such as Rosetta-gami B strains with IPTG induction .

• Subcellular Fractionation: To confirm mitochondrial localization, perform mitochondrial isolation followed by subfractionation to determine if GRPEL1 and its potential interacting partners co-localize in the mitochondrial matrix.

• Proximity Labeling Techniques: Consider BioID or APEX2-based proximity labeling to identify spatial neighbors of GRPEL1 in living cells.

• Fluorescence Microscopy: Use dual-labeling approaches with GRPEL1 antibodies and antibodies against known mitochondrial proteins such as mtHSP70, LONP1, or other potential interactors .

Research has identified several known interactors of GRPEL1, including mtHSP70, LONP1, PP2A B55α, and potentially AIF, which can serve as positive controls in interaction studies .

What protocols yield optimal results for immunofluorescence studies with GRPEL1 antibodies?

For optimal immunofluorescence results with GRPEL1 antibodies, follow these methodological guidelines:

• Cell Preparation:

  • Culture cells on coverslips in appropriate media

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

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

• Antibody Dilution and Incubation:

  • Block with 5% normal serum in PBS for 1 hour

  • Use GRPEL1 antibody at optimal dilution (1:200-1:800)

  • Incubate overnight at 4°C in a humidified chamber

  • Use Alexa Fluor-conjugated secondary antibodies (1:500-1:1000)

• Co-localization Studies:

  • For mitochondrial co-localization, use MitoTracker or co-stain with established mitochondrial markers

  • For ER visualization, consider using pDsRed-ER markers as reference points

  • For live-cell imaging of mitochondria, Mito-DsRed has been successfully used in GRPEL1 studies

• Confocal Imaging Parameters:

  • Use a high-magnification objective (63x or 100x)

  • Acquire z-stack images to ensure complete capture of mitochondrial structures

  • For time-lapse studies, capture images every 2 minutes at 37°C and 5% CO₂

• Controls:

  • Include cells treated with GRPEL1 siRNA (sequence: UUU CGU GGC UGU GCA CAA CAA CCG G) as negative controls

  • Include secondary-only controls to evaluate background fluorescence

HepG2 cells have been validated for GRPEL1 immunofluorescence studies and can serve as positive controls .

What are the critical parameters for successful Western blot detection of GRPEL1?

For optimal Western blot detection of GRPEL1, consider these critical parameters:

• Sample Preparation:

  • Extract total protein using RIPA buffer with protease inhibitors

  • For enriched mitochondrial fractions, consider using differential centrifugation protocols

  • Load 20-50 μg of total protein per lane

• Gel Electrophoresis and Transfer:

  • Use 12-15% SDS-PAGE gels (optimal for 24 kDa proteins)

  • Transfer to PVDF membranes at 100V for 60-90 minutes or 30V overnight at 4°C

• Antibody Incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary GRPEL1 antibody at 1:5000-1:50000 in blocking buffer

  • Incubate overnight at 4°C

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000

• Detection and Visualization:

  • Use enhanced chemiluminescence (ECL) for detection

  • For quantitative analysis, consider fluorescent secondary antibodies and imaging systems

• Controls and Validation:

  • Include GRPEL1 siRNA-treated samples as negative controls

  • MCF-7 and HEK-293T cells are validated positive controls for GRPEL1 detection

  • Expected molecular weight is 24 kDa

• Troubleshooting Tips:

  • If multiple bands appear, optimize antibody dilution or blocking conditions

  • For weak signals, increase protein loading or primary antibody concentration

  • For high background, increase washing steps or reduce antibody concentration

How can GRPEL1 antibodies be used to investigate mitochondrial dysfunction in disease models?

GRPEL1 antibodies can be valuable tools for investigating mitochondrial dysfunction in various disease models through several advanced approaches:

• Tissue-Specific Knockout Models: Research has shown that muscle-specific loss of GRPEL1 causes rapid muscle atrophy, shut down of oxidative phosphorylation, and mitochondrial fatty acid oxidation . Use GRPEL1 antibodies to:

  • Confirm knockout efficiency in different tissues

  • Analyze compensatory expression of related proteins (e.g., GRPEL2)

  • Monitor effects on mitochondrial protein import machinery

• Stress Response Analysis:

  • Investigate ATF4-regulated stress responses following GRPEL1 dysfunction

  • Monitor connections between GRPEL1 depletion and peroxisomal markers like ACOX2

  • Assess integrated stress response activation using GRPEL1 antibodies alongside markers of ER stress and proteotoxic responses

• Inter-organellar Communication Studies:

  • Use GRPEL1 antibodies in combination with markers for peroxisomes, ER, and nucleus to track inter-organellar communication

  • Analyze shifts in TCA cycle intermediates and fatty acid metabolism following GRPEL1 disruption

  • Investigate changes in bile acid regulation as a systemic response

• Clinical Sample Analysis:

  • Research has shown that GrpEL1 levels are seriously compromised in severe dengue virus-infected clinical samples

  • Use immunohistochemistry with GRPEL1 antibodies on patient tissue samples to correlate GRPEL1 levels with disease severity

  • Develop prognostic markers based on GRPEL1 expression patterns

• Cancer Research Applications:

  • Investigate the role of GRPEL1 in the LIV-1-GRPEL1 axis, which has been implicated in cell fate determination during anti-mitotic agent treatment

  • Assess GRPEL1 protein stability and ubiquitination in cancer cells under treatment conditions

What experimental strategies can resolve contradictory data about GRPEL1 function in different model systems?

When facing contradictory data about GRPEL1 function across different model systems, consider these experimental strategies:

• Comprehensive Knockout and Rescue Experiments:

  • Generate complete GRPEL1 knockouts using CRISPR-Cas9

  • Perform rescue experiments with wild-type GRPEL1 and various mutants

  • Use structure-function analysis to map critical domains

  • Compare phenotypes across different cell types and organisms

• Temporal Control of GRPEL1 Expression:

  • Utilize inducible expression systems like tetracycline-inducible lentivirus vectors (as used in previous GRPEL1 studies)

  • Use time-lapse imaging to track cellular responses following GRPEL1 modulation

  • Analyze both immediate and long-term consequences of GRPEL1 loss

• Domainwise Functional Analysis:

  • Create deletion constructs similar to those used in previous studies (aa1-47, aa1-94, aa95-383, aa95-433)

  • Test each construct's ability to rescue GRPEL1-knockout phenotypes

  • Identify cell-type specific interactions that might explain functional differences

• Physiological Context Considerations:

  • Analyze GRPEL1 function under different metabolic states (glycolytic vs. oxidative)

  • Test different stress conditions (oxidative stress, ER stress, proteotoxic stress)

  • Examine cell-type specific cofactors that might influence GRPEL1 function

• Multi-omics Integration:

  • Combine proteomics, transcriptomics, and metabolomics data

  • Look for system-specific compensatory mechanisms

  • Identify model-specific differences in GRPEL1 interaction networks

• In vivo Validation of Key Findings:

  • Verify critical observations from cell culture in animal models

  • Consider tissue-specific conditional knockouts to reconcile differences

How can researchers effectively investigate the role of GRPEL1 in viral pathogenesis?

Research has identified GRPEL1 as a target in viral pathogenesis, particularly in dengue virus infection . To effectively investigate this role, researchers should consider:

• Viral Protease Cleavage Analysis:

  • Perform in vitro cleavage assays using purified components

  • The dengue virus NS3 protease has been shown to cleave GRPEL1 at specific sites (KR₈₁A and QR₉₂S)

  • Use site-directed mutagenesis of these cleavage sites to create cleavage-resistant GRPEL1 variants

• Mitochondrial Protein Import Studies:

  • Investigate how viral proteins (such as dengue NS3pro) are imported into mitochondria

  • Assess whether viral proteins compete with GRPEL1 for import machinery

  • Use purified mitochondria and in vitro import assays to track protein translocation

• Functional Consequences Assessment:

  • Measure mitochondrial functions (respiration, membrane potential) following viral infection

  • Compare effects between wild-type cells and cells expressing cleavage-resistant GRPEL1

  • Investigate whether GRPEL1 cleavage affects mtHsp70 chaperone activity

• Time-Course Experiments:

  • Track GRPEL1 levels throughout viral infection cycles

  • Correlate GRPEL1 cleavage with specific viral replication stages

  • Use time-lapse microscopy with fluorescently tagged GRPEL1 to monitor dynamics

• Clinical Sample Validation:

  • Analyze GRPEL1 levels in clinical samples from patients with varying severity of viral infection

  • Research has shown that GrpEL1 levels are compromised in severe dengue virus-infected clinical samples

  • Correlate GRPEL1 status with clinical outcomes

• Therapeutic Intervention Strategies:

  • Design peptide inhibitors that protect GRPEL1 from viral protease cleavage

  • Test whether preserving GRPEL1 function affects viral replication

  • Investigate whether existing protease inhibitors can prevent GRPEL1 cleavage

How can researchers troubleshoot non-specific binding when using GRPEL1 antibodies?

When encountering non-specific binding with GRPEL1 antibodies, consider these stepwise troubleshooting approaches:

• Antibody Validation and Selection:

  • Verify antibody specificity using GRPEL1 knockdown or knockout samples

  • Consider antibodies validated by enhanced validation techniques such as orthogonal RNAseq

  • For critical applications, compare results from multiple antibodies targeting different GRPEL1 epitopes

• Optimization of Blocking Conditions:

  • Test different blocking agents (BSA, non-fat dry milk, normal serum)

  • Increase blocking duration (2-3 hours at room temperature)

  • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Perform titration experiments with serial dilutions

  • For Western blot, test a wide range (1:5000-1:50000) as recommended

  • For immunofluorescence, optimize between 1:200-1:800

• Pre-absorption Controls:

  • Pre-incubate antibody with immunizing peptide when available

  • Use excess recombinant GRPEL1 protein to absorb specific antibodies

  • Compare patterns before and after pre-absorption

• Cross-Reactivity Analysis:

  • Test antibody against related proteins (especially GRPEL2)

  • Consider potential cross-reactivity with bacterial GrpE proteins in infection models

  • Check reactivity against post-translationally modified GRPEL1

• Application-Specific Optimizations:

  • For Western blot: Increase wash duration/stringency and consider using gradient gels

  • For immunofluorescence: Use confocal microscopy with appropriate controls for autofluorescence

  • For IHC: Test antigen retrieval methods and detection systems

What methodological approaches can differentiate between GRPEL1 and GRPEL2 in experimental systems?

Differentiating between the paralogous proteins GRPEL1 and GRPEL2 requires careful methodological considerations:

• Antibody Selection and Validation:

  • Choose antibodies raised against regions with lowest sequence homology between GRPEL1 and GRPEL2

  • Validate specificity using samples from GRPEL1 or GRPEL2 knockout cells

  • Consider using epitope-tagged versions in overexpression studies

• RNA Interference Approach:

  • Design siRNAs specific to each paralog (e.g., siRNA against GRPEL1: UUU CGU GGC UGU GCA CAA CAA CCG G)

  • Confirm specificity by measuring mRNA levels of both genes after knockdown

  • Use rescue experiments with RNAi-resistant constructs to confirm specificity

• Gene Expression Analysis:

  • Perform quantitative RT-PCR with paralog-specific primers

  • Design primers that span unique exon junctions

  • Use appropriate reference genes for normalization (GAPDH has been used in GRPEL1 studies)

• Functional Differentiation:

  • Research indicates GRPEL1 is essential and cannot be compensated by GRPEL2

  • Study phenotypes in single knockouts of each gene

  • Analyze tissue-specific expression patterns of both paralogs

• Protein-Protein Interaction Profiles:

  • Use immunoprecipitation followed by mass spectrometry to identify unique binding partners

  • Compare interaction networks of GRPEL1 versus GRPEL2

  • Look for differential interactions with mtHsp70 or other mitochondrial proteins

• Subcellular Localization:

  • Perform high-resolution imaging to detect potential differences in submitochondrial localization

  • Use subcellular fractionation followed by Western blotting

  • Consider potential differences in import efficiency or mitochondrial subcompartment targeting

How should researchers interpret changes in GRPEL1 levels in the context of integrated stress responses?

Interpreting changes in GRPEL1 levels during integrated stress responses requires consideration of several factors:

• Multi-Parameter Assessment:

  • Always measure GRPEL1 changes alongside established markers of integrated stress response (ISR) such as ATF4, CHOP, and phosphorylated eIF2α

  • Correlate GRPEL1 changes with mitochondrial functional parameters

  • Consider the temporal sequence of events (immediate vs. delayed responses)

• Transcriptional vs. Post-transcriptional Regulation:

  • Distinguish between changes in GRPEL1 mRNA (using qRT-PCR) and protein levels

  • Assess protein stability through cycloheximide chase experiments

  • Research has shown that GRPEL1 can be regulated post-translationally through ubiquitination

• Organellar Communication Context:

  • Analyze GRPEL1 changes in relation to markers of cross-talk between mitochondria and other organelles

  • Research has identified connections between GRPEL1 dysfunction and peroxisomal responses (e.g., ACOX2 induction)

  • Consider how GRPEL1 changes affect mitochondrial protein import and consequently mitochondrial-nuclear communication

• Tissue-Specific Interpretations:

  • Different tissues show distinct responses to GRPEL1 disruption

  • Muscle-specific loss of GRPEL1 leads to rapid atrophy and metabolic shifts

  • Interpret GRPEL1 changes in the context of tissue-specific metabolic requirements

• Pathological Context Considerations:

  • In viral infections, GRPEL1 can be directly cleaved by viral proteases (e.g., dengue virus NS3)

  • In cancer contexts, GRPEL1 may be regulated through the LIV-1-GRPEL1 axis

  • Different pathological conditions may affect GRPEL1 through distinct mechanisms

• Metabolic Profiling Integration:

  • Correlate GRPEL1 changes with metabolic shifts

  • Research has shown that GRPEL1 disruption can lead to:

    • Fatty acid enrichment in muscle

    • Shifts in TCA cycle intermediates

    • Dysregulated bile acids

Understanding these complex relationships will help researchers accurately interpret GRPEL1 changes in the broader context of cellular stress responses and pathological conditions.

What are the most promising research directions for understanding GRPEL1's role in disease pathogenesis?

Based on current research, several promising directions for investigating GRPEL1 in disease pathogenesis include:

• Cancer Therapeutic Resistance:

  • The LIV-1-GRPEL1 axis has been implicated in determining cell fate during anti-mitotic drug treatment

  • Investigate how GRPEL1 levels correlate with therapeutic responses in different cancer types

  • Explore whether GRPEL1 modulation could sensitize resistant tumors to treatment

• Viral Infection Mechanisms:

  • Further explore how viral proteases (like dengue NS3) target GRPEL1

  • Investigate whether other viruses also target mitochondrial import machinery

  • Develop protective strategies for maintaining GRPEL1 function during viral infection

• Neurodegenerative Diseases:

  • Research has linked GRPEL1 to neurological disease-associated proteins like CHCHD10

  • Investigate GRPEL1's role in maintaining neuronal mitochondrial function

  • Explore potential connections between GRPEL1 dysfunction and protein aggregation disorders

• Metabolic Disorders:

  • GRPEL1 disruption causes shifts in TCA cycle intermediates and dysregulated bile acids

  • Investigate GRPEL1's role in metabolic syndrome and related disorders

  • Explore connections between mitochondrial protein import efficiency and metabolic flexibility

• Muscle Atrophy and Sarcopenia:

  • Muscle-specific loss of GRPEL1 causes rapid muscle atrophy

  • Investigate whether age-related changes in GRPEL1 function contribute to sarcopenia

  • Explore potential therapeutic interventions to maintain GRPEL1 function in aging muscle