GHITM Antibody

What is GHITM and what are its key biological functions?

GHITM, also known as MICS1, TMBIM5, or DERP2, is a mitochondrial protein that localizes to the inner membrane. It plays significant roles in maintaining mitochondrial homeostasis and morphology, particularly in specific cristae structures. GHITM is involved in the apoptotic release of cytochrome c from mitochondria, positioning it as an important regulatory protein in cell death pathways .

Recent research has revealed that GHITM functions in limiting mitochondrial hyperpolarization and reactive oxygen species (ROS) production . Additionally, studies have demonstrated that GHITM can impact the mitochondrial protein synthesis machinery to sustain the structure, shape, and function of mitochondria . These properties make GHITM a critical target for researchers investigating mitochondrial dynamics and cellular stress responses.

Methodologically, when studying GHITM's functions, researchers should consider using both gain-of-function (overexpression) and loss-of-function (knockdown/knockout) approaches to comprehensively characterize its biological effects in specific experimental contexts.

What applications are most suitable for GHITM antibodies?

GHITM antibodies have been validated for multiple research applications, with varying levels of optimization required for each technique:

When selecting the appropriate application, researchers should consider:

• The specific biological question being addressed

• Sample type and availability

• Required sensitivity and specificity

• The need for quantitative (ELISA, WB) versus qualitative or localization data (IHC, IF)

How should samples be prepared for optimal GHITM detection?

Sample preparation varies by sample type and intended application. Follow these methodological guidelines for consistent results:

Serum/Plasma Preparation:

• For serum: Allow samples to clot for 2 hours at room temperature or overnight at 2-8°C

• Centrifuge at approximately 1000 × g (or 3000 rpm) for 15 minutes

• Remove serum and assay immediately or aliquot and store at -20°C or -80°C

Tissue Homogenate Preparation:

• Thoroughly rinse tissues in ice-cold PBS (0.02 mol/L, pH 7.0-7.2)

• Mince tissues into small pieces and homogenize in PBS using a glass homogenizer on ice

• Subject the suspension to ultrasonication or two freeze-thaw cycles to further break down cell membranes

• Centrifuge for 15 minutes at 1500 × g (or 5000 rpm)

• Remove the supernatant for immediate assay or aliquot and store at -20°C or -80°C

Cell Lysate Preparation:

• For adherent cells: Detach with trypsin and collect by centrifugation

• Proceed with appropriate lysis buffer containing protease inhibitors

• For Western blotting applications, include phosphatase inhibitors if phosphorylation status is relevant

IHC Sample Preparation:

• For paraffin-embedded samples, perform antigen retrieval preferably with TE buffer pH 9.0

• Alternatively, use citrate buffer pH 6.0

• Block with 10% normal goat serum for 30 minutes at room temperature

• Incubate with primary antibody (1% BSA) at 4°C overnight

What is the expected molecular weight for GHITM detection in Western blots?

When performing Western blot analysis for GHITM, researchers should expect to observe bands at specific molecular weights depending on the sample type and protein modification status:

• Calculated molecular weight: 37 kDa (based on 345 amino acids)

• Observed molecular weight: primarily 42 kDa, with additional bands at 25-27 kDa

The discrepancy between calculated and observed molecular weights likely results from post-translational modifications or alternative splicing. To ensure specific detection:

• Always include positive controls such as HEK-293 cells, HeLa cells, SH-SY5Y cells, or mouse liver tissue, which have been validated for GHITM expression

• Consider running a gradient gel (8-15%) to improve separation of bands in the 25-45 kDa range

• Validate antibody specificity using GHITM-overexpressing and GHITM-knockdown samples when possible

• For loading controls, consider mitochondrial markers when examining mitochondrial fractions

How should GHITM antibodies be stored and handled?

Proper storage and handling of GHITM antibodies is critical for maintaining their reactivity and specificity:

Storage Conditions:

• Store at -20°C for long-term stability

• Aliquoting is generally unnecessary for -20°C storage for some products

• Avoid repeated freeze/thaw cycles to prevent degradation

Buffer Composition:

• Typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some products may contain 0.1% BSA as a stabilizer

Stability:

• Most GHITM antibodies maintain stability for one year after shipment when stored properly

• Some products have a validated shelf life of 12 months

Handling Recommendations:

• Bring antibody to room temperature before opening

• Gently mix by inverting the tube or gentle pipetting (avoid vortexing)

• Centrifuge briefly before opening to collect contents at the bottom of the tube

• Return to -20°C immediately after use

• Work in a clean environment to prevent contamination

How can GHITM expression analysis inform cancer research, particularly in kidney renal clear cell carcinoma (KIRC)?

GHITM has emerged as a potential biomarker and therapeutic target in kidney renal clear cell carcinoma (KIRC). Recent research has revealed several key aspects of GHITM's role in KIRC that researchers should consider:

Expression Pattern and Clinical Correlation:

• GHITM is downregulated in KIRC compared to normal tissues

• Aberrant GHITM downregulation correlates with clinicopathological features

• Low GHITM expression is associated with unfavorable prognosis in KIRC patients

Diagnostic Value:

• GHITM shows significant diagnostic accuracy with AUC = 0.964 in TCGA-KIRC dataset

• For early-stage (Stage I) patients, GHITM maintained high diagnostic value:

  • AUC = 0.968 in GSE53757 dataset

  • AUC = 0.956 in TCGA-KIRC cohort

Methodological Approaches for GHITM Analysis in KIRC:

• Transcriptomic analysis: Compare GHITM mRNA levels between tumor and adjacent normal tissues using RT-qPCR

• Protein expression analysis: Perform IHC staining on tissue microarrays to correlate GHITM levels with clinicopathological parameters

• Survival analysis: Use Kaplan-Meier curves to evaluate the relationship between GHITM expression and patient outcomes

• ROC curve analysis: Assess GHITM's potential as a diagnostic biomarker for early KIRC detection

When designing studies to investigate GHITM in KIRC or other cancers, researchers should consider both in vitro cell line models and clinical tissue samples to establish comprehensive evidence of its role.

What methodological approaches can be used to study GHITM's effects on tumor cell phenotypes?

Investigating GHITM's effects on tumor cell phenotypes requires a multi-faceted experimental approach. Based on recent research, the following methodological strategies are recommended:

Establishing GHITM-Modified Cell Lines:

• Generate stable GHITM-overexpressing (GHITM-OE) and control (GHITM-EV) cell lines using lentiviral transduction

• Confirm expression levels via Western blot analysis using validated antibodies

Cell Proliferation Assays:

• CCK-8 assay: Measure metabolic activity at 24, 48, 72, and 96 hours post-seeding

• Colony formation assay: Evaluate long-term proliferative capacity (14-21 days)

• EdU incorporation assay: Assess DNA synthesis as a measure of proliferation

Migration and Invasion Assays:

• Wound healing assay: Monitor cell migration into scratch area over 24-48 hours

• Transwell assay: Quantify invasive capacity through Matrigel-coated membranes

Angiogenesis Assessment:

• Tube formation assay: Evaluate the effect of conditioned medium from GHITM-modified cells on endothelial cell tube formation

• Morphological analysis: Observe changes in cell shape and structure using phase-contrast microscopy

In Vivo Tumor Models:

• Subcutaneous xenograft model: Inject GHITM-OE and control cells into immunodeficient mice to assess tumor growth

• Metastasis model: Deliver cells via tail vein injection to evaluate pulmonary metastasis formation

• Tumor analysis: Perform IHC staining of xenografts for proliferation markers (Ki-67) and pathway components (e.g., Notch1)

Recent research has shown that GHITM overexpression inhibits KIRC cell proliferation, migration, and invasion both in vitro and in vivo, supporting its potential tumor-suppressive role .

How can researchers investigate the mechanisms linking GHITM to the Notch signaling pathway?

The connection between GHITM and Notch signaling represents an important area for cancer research. To investigate this relationship, researchers should consider these methodological approaches:

Expression Analysis of Notch Pathway Components:

• Perform Western blot analysis to detect Notch receptors (Notch1-4) in GHITM-overexpressing and control cells

• Use validated antibodies such as:

  • anti-Notch1 (Proteintech, 20687-1-AP)

  • anti-Notch2 (Proteintech, 28580-1-AP)

  • anti-Notch3 (Proteintech, 55114-1-AP)

  • anti-Notch4 (Abcam, ab184742)

Rescue Experiments:

• Co-express GHITM and Notch1 to determine if Notch1 overexpression rescues the phenotypes induced by GHITM upregulation

• Measure endpoints including cell proliferation, migration, and invasion

Notch Pathway Activity Assessment:

• Evaluate expression of Notch target genes using RT-qPCR

• Use luciferase reporter assays with Notch-responsive elements to measure pathway activation

• Assess Notch intracellular domain (NICD) levels by Western blot as an indicator of active Notch signaling

In Vivo Validation:

• Perform IHC staining of xenograft tumors to examine the correlation between GHITM and Notch1 expression

• Analyze human tumor samples for GHITM and Notch pathway component expression patterns

Mechanistic investigations have revealed that GHITM overexpression induces downregulation of Notch1, which acts as an oncogene in KIRC. Furthermore, the inhibitory effects of GHITM upregulation can be effectively rescued by Notch1 overexpression, confirming the functional relationship between these proteins .

What approaches can be used to study GHITM's role in immunotherapy response?

GHITM has recently been identified as a potential modulator of immunotherapy response, particularly related to the PD-1/PD-L1 pathway. Researchers investigating this aspect should consider these methodological strategies:

PD-L1 Expression Analysis:

• Perform Western blot analysis using anti-PD-L1 antibodies (e.g., Proteintech, 28076-1-AP) in GHITM-modified cells

• Conduct flow cytometry to quantify cell surface PD-L1 expression

• Use immunofluorescence to visualize PD-L1 localization patterns

Combination Treatment Studies:

• Evaluate the combined effects of GHITM modulation and PD-1 blockade in preclinical models

• Test GHITM overexpression in combination with other targeted therapies (e.g., sunitinib)

T Cell Co-culture Experiments:

• Co-culture GHITM-modified tumor cells with T cells

• Measure T cell activation markers, proliferation, and cytokine production

• Assess tumor cell killing in the presence of PD-1 blocking antibodies

In Vivo Immunotherapy Models:

• Establish syngeneic tumor models in immunocompetent mice

• Treat with anti-PD-1 antibodies alone or in combination with GHITM modulation

• Monitor tumor growth, survival, and immune infiltration

Recent research has demonstrated that GHITM can regulate PD-L1 protein abundance and that ectopic overexpression of GHITM enhances the antitumor efficiency of PD-1 blockade in KIRC. These findings suggest that GHITM may serve as a valuable target for improving immunotherapy outcomes .

How can researchers investigate transcriptional regulation of GHITM?

Understanding the transcriptional regulation of GHITM is important for comprehensive characterization of its role in normal and disease states. Research has identified YY1 as a transcriptional regulator of GHITM, and the following approaches can be used to study this and other regulatory mechanisms:

Promoter Analysis:

• Perform in silico analysis of the GHITM promoter region to identify potential transcription factor binding sites

• Generate luciferase reporter constructs containing the GHITM promoter region

• Test the effects of candidate transcription factors (e.g., YY1) on reporter activity

Chromatin Immunoprecipitation (ChIP):

• Use ChIP assays to confirm direct binding of transcription factors to the GHITM promoter

• Apply ChIP-seq for genome-wide analysis of transcription factor binding patterns

Transcription Factor Modulation:

• Overexpress or knock down candidate transcription factors (e.g., YY1)

• Measure changes in GHITM mRNA and protein levels

• Use Western blot with anti-GHITM (e.g., Proteintech, 16296-1-AP) and anti-YY1 (e.g., Proteintech, 22156-1-AP) antibodies

Epigenetic Analysis:

• Investigate DNA methylation patterns in the GHITM promoter region

• Examine histone modifications associated with active/repressed transcription

• Test the effects of epigenetic modifying drugs on GHITM expression

Recent research has demonstrated that YY1 can decrease GHITM levels by binding to its promoter. This finding suggests that transcriptional regulation by YY1 may be an important mechanism controlling GHITM expression in cancer contexts .

What are the best approaches for studying GHITM's role in mitochondrial function?

GHITM is known to play a significant role in maintaining mitochondrial homeostasis and morphology. To investigate these functions, researchers should consider these methodological approaches:

Mitochondrial Morphology Analysis:

• Perform live-cell imaging using mitochondrial-specific dyes (e.g., MitoTracker)

• Use confocal microscopy to visualize mitochondrial network structure

• Quantify morphological parameters (length, branching, fragmentation) using specialized software

Mitochondrial Membrane Potential Assessment:

• Use potential-sensitive dyes (e.g., TMRM, JC-1) to measure mitochondrial membrane potential

• Evaluate changes in mitochondrial hyperpolarization in response to GHITM modulation

Reactive Oxygen Species (ROS) Measurement:

• Employ fluorescent probes (e.g., DCFDA, MitoSOX) to quantify cellular and mitochondrial ROS levels

• Assess the impact of GHITM on ROS production under basal and stressed conditions

Cytochrome c Release Assays:

• Fractionate cells into cytosolic and mitochondrial components

• Detect cytochrome c distribution by Western blot

• Evaluate how GHITM modulation affects apoptotic release of cytochrome c

Mitochondrial Protein Synthesis:

• Use pulse-chase labeling with radioactive amino acids to measure mitochondrial protein synthesis rates

• Analyze the composition of respiratory complexes by blue native PAGE

• Assess the impact of GHITM on mitochondrial translation machinery

Previous research has demonstrated that GHITM can limit mitochondrial hyperpolarization and ROS production. Interestingly, in KIRC cell lines, GHITM overexpression did not significantly affect ROS generation or the proportion of apoptotic cells, suggesting context-dependent functions that warrant further investigation .

How can researchers optimize GHITM antibody sensitivity for detecting low expression levels?

When working with samples that may have low GHITM expression levels, researchers can employ several strategies to enhance detection sensitivity:

Western Blot Optimization:

• Increase protein loading (up to 50-80 μg per lane)

• Use higher primary antibody concentration (1:200 dilution)

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

• Switch to more sensitive detection systems (e.g., chemiluminescent substrates with enhanced sensitivity)

• Use PVDF membranes instead of nitrocellulose for improved protein binding

IHC Sensitivity Enhancement:

• Optimize antigen retrieval (TE buffer pH 9.0 is recommended)

• Increase primary antibody concentration (1:20-1:50 dilution)

• Employ signal amplification systems (e.g., tyramide signal amplification)

• Use polymer-based detection systems instead of traditional ABC methods

ELISA Optimization:

• Increase sample volume or concentration

• Extend incubation times

• Optimize buffer conditions

• Use high-sensitivity substrates with extended development time

Sample Enrichment Strategies:

• Perform subcellular fractionation to concentrate mitochondrial proteins

• Use immunoprecipitation to enrich for GHITM before detection

• Consider tissue or cell types with known higher GHITM expression as positive controls

When optimizing detection methods, always include appropriate positive and negative controls to ensure that enhanced sensitivity does not come at the cost of specificity.

What are common pitfalls in GHITM research and how can they be addressed?

Researchers working with GHITM antibodies and studying GHITM function may encounter several challenges. Here are common pitfalls and their solutions:

Antibody Cross-Reactivity:

• Problem: Non-specific binding leading to false-positive signals

• Solution: Validate antibody specificity using GHITM knockout/knockdown controls

• Alternative approach: Use multiple antibodies targeting different epitopes

Inconsistent Molecular Weight Detection:

• Problem: Variable band patterns in Western blots (42 kDa vs. 25-27 kDa)

• Solution: Use gradient gels for better separation and characterize band patterns in positive control samples

• Note: The observed molecular weight (42 kDa) differs from the calculated weight (37 kDa), likely due to post-translational modifications

Mitochondrial Localization Challenges:

• Problem: Difficulty in distinguishing GHITM from other mitochondrial proteins

• Solution: Perform co-localization studies with established mitochondrial markers

• Alternative approach: Use super-resolution microscopy for detailed localization analysis

Context-Dependent Function Interpretation:

• Problem: GHITM's functions may vary across different cell types or disease contexts

• Solution: Include multiple cell lines and primary samples in functional studies

• Alternative approach: Use inducible expression/knockdown systems to study acute versus chronic effects

Reproducibility Across Model Systems:

• Problem: Findings in cell lines may not translate to in vivo models

• Solution: Validate key findings in multiple model systems (cell lines, patient-derived xenografts, clinical samples)

• Alternative approach: Consider species differences when using mouse models, as GHITM antibodies may have different reactivity patterns

By anticipating these challenges and implementing appropriate controls and validation steps, researchers can enhance the reliability and reproducibility of their GHITM-related investigations.