GRAMD4 Antibody

What is GRAMD4 and why is it relevant for cancer research?

GRAMD4 is a 578 amino acid mitochondrial membrane protein that functions as an essential mediator of the p53-independent E2F-1 death pathway. The gene encoding GRAMD4 maps to human chromosome 22, which houses over 500 genes and is the second smallest human chromosome . GRAMD4 plays significant roles in tumor biology through multiple mechanisms:

First, GRAMD4 inhibits tumor metastasis by recruiting the E3 ligase ITCH to promote TAK1 ubiquitination and degradation, leading to the inactivation of MAPK and NF-κB signaling pathways . This mechanism is particularly important in hepatocellular carcinoma (HCC), where GRAMD4 expression is frequently downregulated, and this downregulation predicts worse prognosis for patients after surgical resection .

Second, GRAMD4 functions as a pro-apoptotic protein. Overexpression of GRAMD4 results in a strong apoptotic response involving caspase-3 activation and cleavage of poly(ADP-ribose)-polymerase . This apoptotic function has been observed in multiple cell lines including H1299, Saos-2, and HCT 116 cells .

Third, GRAMD4 has immunomodulatory effects, including inhibition of TLR9 response to nucleic acids and regulation of TLR9-mediated innate immune responses .

What types of GRAMD4 antibodies are available for research use?

Several types of GRAMD4 antibodies are available for research applications, each with specific characteristics:

• Monoclonal antibodies: Products like the PCRP-GRAMD4-1A10 monoclonal antibody target specific epitopes of GRAMD4 and offer high specificity . These are particularly useful for applications requiring consistent lot-to-lot reproducibility.

• Polyclonal antibodies: Products such as ab234649 are rabbit polyclonal antibodies that recognize multiple epitopes within GRAMD4. The immunogen for this particular antibody corresponds to recombinant fragment protein within Human GRAMD4 aa 1-200 . Polyclonal antibodies often provide higher sensitivity but potentially lower specificity compared to monoclonals.

• Region-specific antibodies: Some antibodies target specific regions of GRAMD4, such as the 304-354 amino acid region . These can be valuable for studying particular domains or structural features of the protein.

When selecting an antibody, researchers should consider the specific application (Western blotting, immunohistochemistry, etc.), species reactivity (human, mouse, etc.), and the specific region of GRAMD4 they wish to detect.

What are the validated applications for GRAMD4 antibodies?

GRAMD4 antibodies have been validated for several common laboratory applications:

• Western Blotting (WB): Antibodies like ab234649 have been validated for detecting GRAMD4 in Western blots at dilutions of approximately 1/500. The predicted band size for GRAMD4 is approximately 66 kDa . They have been tested successfully with mouse brain tissue lysate and human cell lines such as MCF7.

• Immunohistochemistry on Paraffin-embedded tissues (IHC-P): GRAMD4 antibodies have demonstrated efficacy in immunohistochemical analysis of paraffin-embedded tissues, typically at dilutions around 1/100 . For example, ab234649 has been successfully used to stain GRAMD4 in human breast cancer tissue.

• Immunofluorescence (IF): Some GRAMD4 antibodies have been utilized in immunofluorescence assays, with dilutions typically around 1:100 .

• Co-Immunoprecipitation (Co-IP): GRAMD4 antibodies have been employed in co-immunoprecipitation experiments to study protein-protein interactions, particularly in investigating the interactions between GRAMD4 and other proteins like TAK1 and ITCH .

When utilizing these applications, researchers should optimize antibody concentrations for their specific experimental conditions and include appropriate positive and negative controls.

How can I design experiments to study GRAMD4's role in tumor suppression?

Designing experiments to investigate GRAMD4's tumor suppressive function requires a multifaceted approach:

How do I troubleshoot inconsistent GRAMD4 antibody staining in tissue samples?

Inconsistent GRAMD4 antibody staining in tissue samples can be addressed through a systematic troubleshooting approach:

• Fixation and processing variables:

  • Standardize fixation protocols using 10% neutral buffered formalin for a consistent duration (24 hours is typical).

  • Ensure consistent tissue processing and paraffin embedding conditions.

  • Consider testing both freshly prepared and archival samples to identify potential degradation issues.

• Antigen retrieval optimization:

  • Compare different antigen retrieval methods (heat-induced vs. enzymatic).

  • Optimize pH conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0).

  • Standardize heating times and cooling periods.

• Antibody validation:

  • Verify antibody specificity using positive control tissues known to express GRAMD4 (liver, heart, or fetal brain tissues are recommended) .

  • Include negative controls (isotype controls or tissues known to lack GRAMD4 expression).

  • Test multiple antibody clones or lots if available.

  • Validate antibody specificity using Western blot of tissue lysates parallel to IHC .

• Titration and incubation parameters:

  • Perform antibody titration experiments (1:50, 1:100, 1:200, etc.).

  • Test different incubation times and temperatures (overnight at 4°C vs. 1-2 hours at room temperature).

  • Optimize blocking conditions to reduce background staining.

• Detection system considerations:

  • Compare different detection systems (polymer-based vs. avidin-biotin).

  • Evaluate chromogen development times.

  • Consider signal amplification methods for low-abundance targets.

• Technical controls:

  • Implement multi-tissue controls on every slide.

  • Process all experimental samples simultaneously to minimize batch effects.

  • Document all experimental conditions thoroughly for reproducibility.

How can I study the relationship between GRAMD4 and TAK1 in cancer progression?

Investigating the GRAMD4-TAK1 relationship in cancer progression requires sophisticated experimental approaches:

What are the optimal protocols for detecting GRAMD4 in different sample types?

Optimizing GRAMD4 detection across different sample types requires specific methodological considerations:

For Western Blotting:

• Sample preparation:

  • For tissues: Homogenize in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors.

  • For cells: Lyse in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS with protease inhibitors .

  • Include sonication step to shear genomic DNA and reduce sample viscosity.

• Electrophoresis and transfer conditions:

  • Load 20-50 μg protein per lane on 10% SDS-PAGE gels.

  • Use wet transfer at 100V for 90 minutes with chilled transfer buffer containing 20% methanol.

  • Verify transfer efficiency with reversible protein stains like Ponceau S.

• Antibody incubation:

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

  • Incubate with primary GRAMD4 antibody at 1:500 dilution in 5% BSA in TBST overnight at 4°C .

  • Wash extensively with TBST (3 × 10 minutes).

  • Incubate with secondary antibody (goat anti-rabbit IgG-HRP) at 1:50,000 dilution for 1 hour at room temperature .

  • Develop using enhanced chemiluminescence detection.

For Immunohistochemistry:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin for 24 hours.

  • Process and embed in paraffin following standard protocols.

  • Section at 4-5 μm thickness onto positively charged slides.

• Staining protocol:

  • Deparaffinize sections and perform heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes.

  • Block endogenous peroxidase with 3% H₂O₂ in methanol.

  • Apply protein block (5% normal goat serum) for 30 minutes.

  • Incubate with GRAMD4 antibody at 1:100 dilution overnight at 4°C .

  • Apply appropriate secondary antibody and develop with DAB chromogen.

  • Counterstain with hematoxylin, dehydrate, and mount.

• Scoring system:

  • Evaluate staining based on percentage of positive cells (0: <10%, 1: 10-25%, 2: 26-50%, 3: 51-75%, 4: >75%) and intensity (0: negative, 1: light brown, 2: brown, 3: dark brown) .

  • Calculate total score by multiplying these values, with scores 0-5 defined as negative and 6-12 as positive .

How should I validate GRAMD4 antibody specificity for my research?

Rigorous validation of GRAMD4 antibody specificity is essential for generating reliable research data:

• Positive and negative controls:

  • Include known positive controls: human liver, heart, or fetal brain tissues are recommended for GRAMD4 antibody validation .

  • Include negative controls: tissues known to express minimal GRAMD4 or tissues from GRAMD4 knockout models if available.

  • Use isotype controls to identify non-specific binding.

• Cross-platform validation:

  • Compare antibody detection across multiple techniques (WB, IHC, IF) to ensure consistent target recognition .

  • For Western blotting, verify that the detected band matches the predicted molecular weight of GRAMD4 (approximately 66 kDa) .

  • For IHC/IF, verify that subcellular localization is consistent with known GRAMD4 distribution (mitochondrial membrane) .

• Genetic validation approaches:

  • Perform antibody testing in GRAMD4 knockdown models (using shRNAs or CRISPR/Cas9) .

  • Compare detection in GRAMD4 overexpression systems versus empty vector controls .

  • Include rescue experiments to confirm antibody specificity.

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide or recombinant GRAMD4 protein.

  • Loss of signal in subsequent detection assays confirms specificity for the target epitope.

• Cross-reactivity assessment:

  • Test the antibody against related family members or proteins with similar domains.

  • Evaluate species cross-reactivity if working with multiple model organisms.

  • Document any potential cross-reactivity in experimental reports.

• Lot-to-lot validation:

  • Perform side-by-side comparisons of different antibody lots.

  • Maintain detailed records of validation results for each lot.

  • Consider creating an internal reference standard for long-term projects.

What are the best approaches for quantifying GRAMD4 expression in cancer samples?

Accurate quantification of GRAMD4 expression in cancer samples requires appropriate methodological approaches:

• Immunohistochemical quantification:

  • Utilize digital pathology platforms for objective quantification of GRAMD4 staining.

  • Apply the established scoring system that considers both percentage of positively stained cells (0-4) and staining intensity (0-3), with total scores calculated by multiplication .

  • Include multiple fields per sample (minimum 5) to account for tumor heterogeneity.

  • Perform scoring by at least two independent pathologists blinded to clinical data, with discrepancies resolved by consensus .

• mRNA expression analysis:

  • Extract RNA from fresh-frozen or FFPE tumor samples using optimized protocols.

  • Perform RT-qPCR with validated GRAMD4-specific primers and appropriate reference genes.

  • Consider utilizing data from public repositories such as TCGA, GEO (GSE14520, GSE22058, GSE63898), or proteomics data from CPTAC .

  • Normalize expression using multiple housekeeping genes selected for stability in the tissue type being studied.

• Western blot quantification:

  • Include loading controls appropriate for the sample type (β-actin, GAPDH, vinculin).

  • Utilize LI-COR Odyssey or similar systems that offer a wider linear range than chemiluminescence.

  • Perform densitometric analysis with appropriate software (ImageJ, Image Studio Lite).

  • Present data as normalized ratios relative to loading controls and calibrator samples.

• Multi-omics integration:

  • Correlate protein expression data with mRNA expression, copy number variations, and methylation status .

  • Analyze GRAMD4 expression in the context of related pathway components (TAK1, ITCH, MAPK, NF-κB) .

  • Consider single-cell approaches to address tumor heterogeneity.

  • Develop multivariate models incorporating GRAMD4 expression and other molecular features to predict clinical outcomes .

• Statistical considerations:

  • Apply appropriate statistical tests based on data distribution (parametric vs. non-parametric).

  • Correct for multiple testing when performing genome-wide comparisons.

  • Include power calculations to ensure adequate sample sizes.

  • Consider survival analysis methods like Cox proportional hazards models to assess the prognostic significance of GRAMD4 expression .