GNG5 Antibody

What is GNG5 and what cellular functions does it mediate?

GNG5 is a subunit of heterotrimeric G proteins involved in transmembrane signaling systems. It functions as a modulator or transducer in various signaling pathways. The beta and gamma chains are required for GTPase activity, for replacement of GDP by GTP, and for G protein-effector interactions . Recent research has established GNG5 as an oncogene in several cancer types, including gliomas, lymphoma, and metastatic thymic adenocarcinoma .

What types of GNG5 antibodies are available for research applications?

Several types of GNG5 antibodies are available for research, with the most common being rabbit polyclonal antibodies. For example, ab238835 is a rabbit polyclonal antibody suitable for immunohistochemistry on paraffin-embedded tissues (IHC-P) and immunocytochemistry/immunofluorescence (ICC/IF) applications with human samples . Other options include product 15336-1-AP, which targets GNG5 in ELISA applications and shows reactivity with human, mouse, and rat samples .

What is the molecular weight of the GNG5 protein and how does this affect antibody selection?

GNG5 has a calculated molecular weight of approximately 7 kDa . This small size is important to consider when selecting antibodies and designing experiments, particularly for Western blot applications. When running protein gels for GNG5 detection, use higher percentage gels (10-15%) to properly resolve these small molecular weight proteins. Additionally, transfer conditions may need optimization for efficient transfer of small proteins to membranes .

What are the recommended storage conditions for GNG5 antibodies?

Most GNG5 antibodies should be stored at -20°C and remain stable for one year after shipment. For example, the 15336-1-AP antibody is stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Aliquoting is generally unnecessary for -20°C storage, and some preparations (like 20μl sizes) may contain 0.1% BSA for stability . Always check manufacturer specifications for particular antibodies to ensure optimal storage conditions.

What are the validated applications for GNG5 antibodies in cancer research?

GNG5 antibodies have been validated for multiple applications in cancer research, including immunohistochemistry on paraffin-embedded tissues (IHC-P), immunocytochemistry/immunofluorescence (ICC/IF), Western blotting, and ELISA . These antibodies have been successfully applied to study GNG5 expression in various cancer types including glioma, hepatocellular carcinoma, and breast cancer . Applications include detecting differential expression between tumor and normal tissues, evaluating subcellular localization, and assessing correlation with clinical outcomes.

What protocol modifications are recommended for Western blot detection of GNG5?

For optimal Western blot detection of GNG5, researchers should use the following protocol modifications:

• Use 10% SDS-PAGE gels to properly separate this small protein (7 kDa)

• Transfer onto PVDF membranes

• Block with 5% defatted milk containing Tris-buffered saline with 0.1% Tween 20

• Incubate with primary antibody (typical dilution 1:5000) at 4°C

• Follow with appropriate horseradish peroxidase-labeled secondary antibody (typical dilution 1:10000)

For normalization, GAPDH is commonly used as a loading control . Verification of GNG5 knockdown efficiency in functional studies typically requires Western blotting to confirm greater than 50% protein reduction .

How can I optimize immunohistochemistry protocols for GNG5 detection in tissue samples?

For optimal immunohistochemistry staining of GNG5 in tissue samples, researchers should use antibodies validated for IHC-P applications such as ab238835 or Atlas Antibodies Cat#HPA043651 . Typically, these antibodies work best at dilutions around 1:100 for paraffin-embedded tissues. Successful staining has been demonstrated in various tissues including liver cancer tissue, kidney tissue, and breast cancer samples . Counterstaining methods and antigen retrieval techniques should be optimized based on the specific tissue type being examined.

How can GNG5 expression analysis be integrated with immune infiltration studies?

Researchers can analyze the correlation between GNG5 expression and immune characteristics using several computational approaches. The TIMER database (https://cistrome.shinyapps.io/timer/) can be used to analyze the infiltration of immune cells in tumor tissues with specific RNA-Seq expression profile data. The CIBERSORT algorithm can assess abundance differences of immune cells between high and low GNG5 expression groups .

Studies have shown that GNG5 expression positively correlates with several immune cell types, including Th2 cells, TFH, macrophages, aDC, Th1 cells, helper T cells, iDC, NK CD56bright cells, and B cells . These correlations should be considered when designing experiments that examine the role of GNG5 in the tumor microenvironment and immune responses.

What signaling pathways are affected by GNG5 modulation in cancer cells?

Research indicates that GNG5 significantly influences several crucial signaling pathways in cancer. In breast cancer, GNG5 achieves cell growth and invasion through activation of the Wnt/β-catenin pathway . After GNG5 knockdown, expression changes in key proteins including β-catenin, cyclin D1, and c-myc have been observed.

For researchers investigating signaling mechanisms, Gene Set Enrichment Analysis (GSEA) can help identify GNG5-related pathways by using expression level of GNG5 as a reference phenotype tag. Enriched pathways can be analyzed based on nominal (NOM) P-values and normalized enrichment scores (NES) . This approach allows for comprehensive mapping of the molecular networks influenced by GNG5 modulation.

How reliable is GNG5 as a prognostic biomarker across different cancer types?

For hepatocellular carcinoma, researchers have compiled data from multiple databases (TCGA, GTEx, ICGC) to evaluate GNG5's prognostic value . When conducting similar analyses, researchers should consider using multivariate Cox regression analysis to determine whether GNG5 is an independent prognostic factor when accounting for other clinical variables such as tumor stage, grade, and established biomarkers.

What are the recommended approaches for GNG5 knockdown in functional studies?

For effective GNG5 knockdown in functional studies, short hairpin RNA (shRNA) approaches have proven successful. In previous studies, sh-GNG5 constructs have effectively knocked down more than 50% of GNG5 expression in cancer cell lines such as BT549 and MDA-MB-231 . Researchers should design multiple shRNA constructs targeting different regions of the GNG5 mRNA to identify the most effective knockdown strategy.

Verification of knockdown efficiency should be performed using both quantitative RT-PCR and Western blot to confirm reduction at both mRNA and protein levels. For RT-qPCR analysis, GAPDH is commonly used for normalization of GNG5 expression . After confirming knockdown, functional assays including cell proliferation (CCK-8), clone formation, migration (Transwell), and invasion assays can be performed to assess the biological effects of GNG5 silencing.

What phenotypic changes are expected after GNG5 knockdown in cancer cells?

Based on previous research, GNG5 knockdown in cancer cells typically results in several phenotypic changes:

• Reduced cell proliferation as measured by CCK-8 assay

• Decreased clone formation capacity

• Suppressed cell migration and invasion capabilities

• Altered expression of epithelial-mesenchymal transition (EMT) markers, including upregulation of E-cadherin and downregulation of N-cadherin

How can cross-reactivity issues with GNG5 antibodies be addressed?

Cross-reactivity can be a concern with GNG5 antibodies due to the presence of related G protein subunits. To address this issue, researchers should:

• Select antibodies specifically validated for the species being studied. For example, some antibodies like 15336-1-AP have been tested for reactivity with human, mouse, and rat samples .

• Include appropriate controls in experiments, including positive control tissues known to express GNG5 and negative controls with GNG5 knocked down.

• Validate antibody specificity using Western blot to confirm detection of a single band at the expected molecular weight (7 kDa).

• Consider using antibodies raised against recombinant full-length protein corresponding to the target species for improved specificity .

If cross-reactivity is detected, optimization of antibody dilution and incubation conditions may help improve specificity.

What are the most common technical challenges when analyzing GNG5 expression in clinical samples?

When analyzing GNG5 expression in clinical samples, researchers frequently encounter several technical challenges:

• Tissue heterogeneity: Variations in cell type composition can affect expression analysis. Using techniques like laser capture microdissection can help isolate specific cell populations.

• Sample preservation effects: RNA and protein quality may be affected by fixation methods and storage duration. Standardizing collection protocols is essential.

• Quantification standardization: For immunohistochemistry analysis, implementing standardized scoring systems helps reduce inter-observer variability.

• Reference gene selection: When performing RT-qPCR analysis, appropriate reference genes (such as GAPDH) must be selected for accurate normalization .

For clinical sample analysis, it's advisable to use multiple detection methods (e.g., combining RT-qPCR with immunohistochemistry) to strengthen findings and account for technique-specific limitations.

How can researchers effectively combine GNG5 analysis with multi-omics approaches?

To effectively integrate GNG5 analysis with multi-omics approaches, researchers should:

• Design experiments that collect matched samples for different omics analyses (transcriptomics, proteomics, etc.)

• Utilize bioinformatics tools for integrative analysis, such as co-expression analysis and GSEA to detect GNG5-related genes and possible signaling pathways

• Cross-validate findings using multiple databases like TCGA, GTEx, and ICGC

• Apply computational methods such as ESTIMATE to evaluate immune scores, tumor purity, and stromal scores in relation to GNG5 expression

• Consider single-cell approaches to resolve heterogeneity issues in complex samples

This integrated approach provides a more comprehensive understanding of GNG5's role in biological systems and its potential as a biomarker or therapeutic target.