GRPEL2 Antibody

What is GRPEL2 and what are its key biological functions?

GRPEL2 is a mitochondrial protein encoded by the GRPEL2 gene that functions as a nucleotide exchange factor. Based on recent experimental evidence, GRPEL2 plays a significant role in cellular processes including regulation of cell growth, metastasis, and apoptosis. Research findings demonstrate that GRPEL2 knockdown leads to suppressed cell growth and metastasis while promoting cell apoptosis in hepatocellular carcinoma models . These effects appear to be mediated through its regulation of cell cycle progression, reactive oxygen species (ROS) production, and mitochondrial membrane potential (MMP) . The protein has been identified as a potential oncogenic factor in HCC development, suggesting its importance in cellular proliferation pathways.

What species reactivity can be expected when using GRPEL2 antibodies?

GRPEL2 antibodies demonstrate variable cross-reactivity depending on the specific antibody and its binding region. Current commercially available GRPEL2 antibodies show reactivity with multiple species:

• Antibodies targeting the middle region of GRPEL2 (e.g., ABIN2785166) demonstrate broad reactivity across species including human, mouse, rat, dog, guinea pig, horse, rabbit, cow, and zebrafish (Danio rerio) .

• Antibodies targeting amino acids 33-225 (e.g., ABIN6141437) show more limited reactivity with human, mouse, and rat specimens .

The predicted reactivity percentages for middle region targeting antibodies are:

• Human: 100%

• Mouse: 100%

• Rat: 100%

• Dog: 100%

• Guinea Pig: 100%

• Horse: 100%

• Rabbit: 100%

• Cow: 93%

• Zebrafish: 79%

This cross-reactivity profile enables comparative studies across multiple model organisms when investigating GRPEL2 function.

What are the common applications for GRPEL2 antibodies in research settings?

GRPEL2 antibodies serve multiple applications in research settings, with validated methodologies for:

• Western Blotting (WB): Most commercially available GRPEL2 antibodies have been validated for western blotting applications. For instance, the ABIN2785166 antibody was validated using cell lysates as positive controls . This technique allows researchers to detect and quantify GRPEL2 protein expression levels in various cell and tissue samples.

• Immunohistochemistry (IHC): Several GRPEL2 antibodies, including ABIN6141437, are suitable for IHC applications . In HCC research, IHC has been performed on tissue microarrays using anti-GRPEL2 antibodies (e.g., ab229985) to evaluate expression patterns . The protocol typically involves:

  • Deparaffinization and rehydration of tissue

  • Peroxidase blocking (28°C for 25 min)

  • Antigen retrieval in pH 6.0 citrate buffer (28°C for 30 min)

  • Blocking with 5% BSA in PBS (1 h at 28°C)

  • Primary antibody incubation (4°C overnight)

  • Secondary antibody incubation (28°C for 30 min)

  • HRP-conjugated streptavidin incubation (28°C for 15 min)

  • DAB staining for signal determination

• ELISA: Some antibodies are validated for ELISA applications, enabling quantitative measurement of GRPEL2 in solution.

These applications provide researchers with methodological options to investigate GRPEL2 expression and function in various experimental contexts.

How should GRPEL2 antibody staining patterns be interpreted in immunohistochemistry?

Interpreting GRPEL2 antibody staining patterns in immunohistochemistry requires both qualitative assessment and semi-quantitative scoring. Based on research protocols:

• Qualitative assessment: GRPEL2 typically demonstrates cytoplasmic staining patterns, consistent with its mitochondrial localization. Researchers should examine subcellular distribution patterns to confirm appropriate localization.

• Semi-quantitative scoring: The German semi-quantitative scoring system has been validated for GRPEL2 expression analysis, scoring staining intensity as:

  • No staining = 0

  • Weak staining = 1

  • Moderate staining = 2

  • Strong staining = 3

• Expression level quantification: GRPEL2 expression can be semi-quantified using the formula:
  Expression level = intensity score × positive rate × 100

For accurate interpretation, image capture using panoramic scanners (such as PANNORAMIC 3DHISTECH) and analysis with appropriate software (such as CaseViewer 2.4 or AIpathwell) is recommended for standardized evaluation . Researchers should establish consistent scoring criteria across specimens and ensure blinded evaluation to minimize bias.

What controls should be included when validating GRPEL2 antibodies?

Proper validation of GRPEL2 antibodies requires inclusion of several critical controls:

• Positive controls:

  • Cell lysates with known GRPEL2 expression for Western blotting

  • Tissue sections with documented GRPEL2 expression for IHC (such as liver tissue)

  • Recombinant GRPEL2 protein for antibody specificity testing

• Negative controls:

  • Primary antibody omission control to assess non-specific binding of secondary antibodies

  • Isotype controls using non-specific IgG matching the host species and concentration of the primary antibody

  • Tissues known to lack GRPEL2 expression

• Knockdown/knockout validation:

  • Samples from GRPEL2 knockdown experiments using specific shRNAs (such as shGRPEL2-1 and shGRPEL2-2)

  • CRISPR/Cas9-mediated GRPEL2 knockout cells or tissues

• Peptide competition:

  • Pre-incubation of antibody with the immunizing peptide to demonstrate binding specificity

These controls collectively ensure antibody specificity, minimize false positives, and validate experimental findings in GRPEL2 research.

What methodological approaches are optimal for investigating GRPEL2's role in oncogenic pathways?

Investigating GRPEL2's role in oncogenic pathways requires a multi-faceted experimental approach combining genetic, biochemical, and functional analyses:

• Genetic manipulation strategies:

  • Lentiviral-mediated shRNA knockdown: Utilizing lentiviral plasmids (such as pLKO.1-TRC) to express shRNAs targeting GRPEL2. This approach has been successful with constructs like shGRPEL2-1 and shGRPEL2-2 .

  • Overexpression systems: Employing vectors like pCDH-CMV-MCS-T2A-puro for GRPEL2 overexpression studies in cell lines such as Hep3B .

  • CRISPR/Cas9-mediated gene editing for complete knockout studies.

• Functional assays to assess oncogenic mechanisms:

  • Cell proliferation: MTT or BrdU incorporation assays to measure growth effects

  • Apoptosis analysis: Flow cytometry with Annexin V/PI staining

  • Migration and invasion: Transwell assays to assess metastatic potential

  • Colony formation: Evaluating clonogenicity following GRPEL2 manipulation

  • Cell cycle analysis: PI staining with flow cytometry to detect cell cycle distribution changes

• Pathway analysis:

  • Gene Set Enrichment Analysis (GSEA): Proven effective in identifying pathways affected by GRPEL2, including cell cycle and NF-κB signaling .

  • Pharmacological inhibitor studies: Using pathway-specific inhibitors (such as PDTC for NF-κB pathway) to validate mechanism involvement .

  • Western blotting for pathway components: Assessing activation status of NF-κB and cell cycle regulators.

• In vivo validation:

  • Xenograft models using GRPEL2-manipulated cell lines to confirm in vitro findings

  • Analysis of tumor growth, metastasis, and survival outcomes

This comprehensive approach enables researchers to establish causality and mechanistic details of GRPEL2's role in oncogenic pathways.

How can researchers address potential cross-reactivity issues when using GRPEL2 antibodies in multi-species studies?

Addressing cross-reactivity issues in multi-species GRPEL2 antibody studies requires systematic validation approaches:

• Epitope sequence analysis:

  • Compare the epitope sequences across target species using sequence alignment tools

  • The middle region epitope (HEHELICHVP AGVGVQPGTV ALVRQDGYKL HGRTIRLARV EVAVESQRRL) shows high conservation across species

  • Predicted reactivity varies from 79% (zebrafish) to 100% (human, mouse, etc.)

  • Select antibodies based on predicted sequence homology for multi-species studies

• Validation strategy for cross-species applications:

  • Perform western blot validation in tissues from each species of interest

  • Include positive controls from species with known reactivity

  • Run comparative immunoprecipitation followed by mass spectrometry to confirm target specificity

  • Use antibodies targeting different epitopes to confirm findings

• Epitope-specific considerations:

  • Middle region antibodies (e.g., ABIN2785166) show broader cross-reactivity

  • Amino acid-specific antibodies (e.g., AA 33-225, ABIN6141437) demonstrate more limited cross-reactivity

  • C-terminal antibodies have different reactivity profiles than N-terminal antibodies

• Alternative approaches when cross-reactivity is problematic:

  • Species-specific antibody development using unique epitope regions

  • Molecular techniques (qPCR for mRNA expression) as complementary approaches

  • Epitope tagging of GRPEL2 in model organisms followed by tag-specific antibody detection

This systematic approach ensures reliable cross-species comparisons while minimizing false positive or negative results due to antibody cross-reactivity issues.

What molecular mechanisms underlie GRPEL2's effects on mitochondrial function and cell survival?

GRPEL2's effects on mitochondrial function and cell survival involve several interconnected molecular mechanisms:

These mechanisms collectively contribute to GRPEL2's effects on cell proliferation, apoptosis, and oncogenic potential, providing multiple intervention points for targeted therapies.

How should researchers design experiments to investigate the differential roles of GRPEL2 in normal versus malignant tissues?

Designing experiments to investigate differential GRPEL2 roles in normal versus malignant tissues requires a comprehensive approach:

This experimental framework enables systematic comparison of GRPEL2's normal physiological functions versus its pathological roles in malignancy.

What technical considerations are important when using GRPEL2 antibodies for detecting low-abundance targets?

Detecting low-abundance GRPEL2 requires specialized technical considerations across multiple methods:

• Western blotting optimization for low-abundance detection:

  • Sample preparation enhancement:

    • Implement mitochondrial enrichment procedures to concentrate GRPEL2

    • Utilize RIPA or NP-40 buffers with protease inhibitor cocktails

    • Increase protein loading (50-100 μg total protein)

  • Detection sensitivity improvement:

    • Use high-sensitivity chemiluminescent substrates

    • Implement signal amplification systems

    • Consider biotin-streptavidin based detection systems

  • Antibody protocol optimization:

    • Extend primary antibody incubation to overnight at 4°C

    • Titrate antibody concentrations to determine optimal signal-to-noise ratio

    • Consider signal enhancement systems like tyramide signal amplification

• Immunohistochemistry adaptations:

  • Antigen retrieval enhancement:

    • Extended citrate buffer treatment (pH 6.0) at optimal temperature (28°C for 30 min)

    • Consider alternative retrieval methods for comparison

  • Signal amplification:

    • Implement polymer-based detection systems

    • Utilize HRP-conjugated streptavidin systems (15 min incubation)

    • Consider catalyzed reporter deposition methods

  • Background reduction:

    • Extended blocking (5% BSA, 1 hour at 28°C)

    • Add protein-based blockers to antibody diluents

    • Include stringent washing steps

• Fluorescence-based detection approaches:

  • Fluorophore selection:

    • Use bright, photostable fluorophores

    • Consider quantum dots for stable signal

  • Instrumentation considerations:

    • Utilize confocal microscopy for improved signal isolation

    • Implement spectral unmixing to distinguish signal from autofluorescence

  • Image acquisition optimization:

    • Extend exposure times within linear range

    • Implement signal averaging across multiple acquisitions

    • Use deconvolution algorithms to enhance signal

• Validation and controls:

  • Comparative analysis against overexpression systems

  • GRPEL2 knockdown controls to establish detection limits

  • Serial dilution curves to determine limit of detection

These technical considerations collectively maximize sensitivity while maintaining specificity when detecting low-abundance GRPEL2 protein.