GNG12 Antibody

What is GNG12 and what are its key biological functions?

GNG12 (G Protein subunit gamma 12) is a member of the G protein family that acts as a modulator in various transmembrane signaling pathways. It consists of 72 amino acids with a calculated molecular weight of approximately 8 kDa . This protein participates in:

• Modulation of inflammatory signaling cascades

• Regulation of interactions between Ca²⁺ and cyclic AMP through protein kinase C-dependent phosphorylation

• Blocking inflammatory responses induced by lipopolysaccharide

• Activation of mammalian target of rapamycin signaling to promote cell proliferation

Recent research has revealed GNG12's significant role in cancer biology, particularly in pancreatic ductal adenocarcinoma (PDAC), where it promotes cancer cell growth and modulates immune responses through NF-κB signaling pathway activation .

What applications are GNG12 antibodies typically used for in research?

GNG12 antibodies are versatile tools in molecular and cellular biology research with multiple applications:

The selection of application depends on the specific experimental goals, sample type, and required sensitivity level .

What are important considerations when selecting a GNG12 antibody for experimental use?

When selecting GNG12 antibodies for research applications, several critical factors should be considered:

• Species reactivity: Different antibodies show specific reactivity patterns. For example, ABIN7139863 is reactive with human samples only, while 15781-1-AP shows reactivity with human, mouse, and rat samples .

• Binding specificity: Consider the epitope region. Some antibodies target specific amino acid regions of GNG12:

  • AA 2-17 (ABIN7139863)

  • AA 37-68 (ABIN6243817)

  • AA 25-55 (other variants)

• Clonality: Most available GNG12 antibodies are polyclonal, which offers broader epitope recognition but may introduce batch-to-batch variability .

• Host species: Typically produced in rabbit, which is important to consider when designing multi-color immunostaining experiments to avoid cross-reactivity .

• Validation data: Review available validation information. For instance, several antibodies have validation data for specific applications as indicated in product information sheets .

• Storage conditions: Most GNG12 antibodies require storage at -20°C and are stable for one year after shipment .

How can GNG12 antibodies be utilized to investigate its role in cancer pathology, particularly in pancreatic ductal adenocarcinoma?

GNG12 has emerged as a significant player in pancreatic ductal adenocarcinoma (PDAC) pathology. Research approaches using GNG12 antibodies can include:

• Expression profiling in clinical specimens:

  • Immunohistochemistry (IHC) has revealed higher GNG12 expression in PDAC patient specimens compared to nontumor pancreatic tissues .

  • Method: Use GNG12 antibodies (such as ab154698 at 1:150 dilution) for IHC on tissue microarrays (TMA) .

  • Scoring: Implement blinded evaluation where 1 = weak staining at ×100 magnification; 2 = medium staining at ×40 magnification; 3 = strong staining at ×40 magnification .

• Correlation with prognostic factors:

  • High GNG12 expression correlates with poor prognosis in PDAC patients .

  • Analytical tools include Oncomine web tool, GEPIA (gene expression profiling interactive analysis), and Human Protein Atlas for survival analysis .

• Mechanistic studies on NF-κB signaling activation:

  • Western blotting using GNG12 antibodies (typically at 1:1000 dilution) can be combined with p65 antibodies to analyze nuclear translocation of p65 as an indicator of NF-κB pathway activation .

  • qRT-PCR can be used to measure changes in NF-κB target gene expression (TNF, IL-1α, IL-6, CD83, GADD45B, BCL2L1, CXCL5, CXCR1, FOS, NR4A2) following GNG12 manipulation .

• PD-L1 regulation studies:

  • Combination of GNG12 and PD-L1 antibodies in Western blot and qRT-PCR analyses can reveal the relationship between GNG12 and immune checkpoint regulation .

What experimental approaches can be used to validate GNG12 antibody specificity and overcome potential cross-reactivity issues?

Ensuring antibody specificity is crucial for obtaining reliable research results. For GNG12 antibodies, consider these validation approaches:

• Genetic knockdown/knockout controls:

  • Use lentivirus vector-based short hairpin RNAs (shRNAs) targeting GNG12 as described in research by Li et al.

  • Western blotting with GNG12 antibody should show reduced signal in knockdown samples .

• Overexpression systems:

  • Use Flag-GNG12 vector (pcDNA3.1 backbone mixed with GNG12 cDNA) to create overexpression controls .

  • Verify increased signal with GNG12 antibody in transfected vs. non-transfected cells.

• Multiple antibody validation:

  • Test multiple GNG12 antibodies targeting different epitopes to confirm consistent staining patterns.

  • Compare results from commercially available antibodies such as ab154698 (Abcam) and 15781-1-AP (Proteintech) .

• Peptide competition assay:

  • Pre-incubate the GNG12 antibody with the immunizing peptide before application.

  • Signal should be reduced or eliminated if antibody is specific.

• Mass spectrometry validation:

  • Immunoprecipitate GNG12 using the antibody and confirm protein identity by mass spectrometry.

How do experimental conditions affect GNG12 antibody performance in different applications?

Optimizing experimental conditions is essential for successful GNG12 antibody applications:

• Western blotting optimization:

  • Sample preparation: Use appropriate lysis buffers that preserve G protein integrity.

  • Protein denaturation: GNG12 (8 kDa) requires careful denaturation conditions to avoid protein aggregation or degradation.

  • Gel percentage: Use higher percentage gels (15-20%) for optimal resolution of this small protein.

  • Transfer conditions: Adjust transfer time and voltage for efficient transfer of small proteins.

  • Blocking: 5% non-fat milk or BSA in TBST, typically for 1 hour at room temperature.

  • Primary antibody incubation: Dilutions range from 1:1000 to 1:5000, optimally incubated overnight at 4°C .

• Immunohistochemistry considerations:

  • Fixation: Formalin-fixed, paraffin-embedded tissues require antigen retrieval methods.

  • Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0).

  • Antibody dilution: For GNG12 antibodies, recommended dilutions range from 1:20 to 1:200 .

  • Incubation time: Typically overnight at 4°C for primary antibody.

  • Detection systems: Amplification systems may improve sensitivity for low-expressing samples.

• Immunofluorescence optimization:

  • Fixation: 4% paraformaldehyde for 15-20 minutes at room temperature.

  • Permeabilization: 0.1-0.5% Triton X-100 for 5-10 minutes.

  • Blocking: 1-5% BSA or normal serum from the secondary antibody host species.

  • Primary antibody: Dilutions typically range from 1:50 to 1:200 .

  • Co-staining considerations: When performing double or triple immunofluorescence, consider antibody compatibility.

What methodological approaches can be used to investigate GNG12's role in modulating immune responses through NF-κB signaling?

Research has established GNG12's role in regulating immune responses through NF-κB signaling, particularly in cancer contexts. Several methodological approaches can be employed:

• Subcellular fractionation and nuclear translocation assays:

  • Separate nuclear and cytoplasmic fractions from cells with manipulated GNG12 expression.

  • Use Western blotting with p65 antibodies (1:1000 dilution) to detect nuclear translocation of p65 as evidence of NF-κB activation .

  • Include proper loading controls (GAPDH for cytoplasmic fraction, nuclear-specific markers like Lamin B for nuclear fraction).

• Luciferase reporter assays:

  • Transfect cells with an NF-κB responsive element-driven luciferase reporter.

  • Measure luciferase activity in cells with GNG12 knockdown or overexpression.

  • Include positive controls (TNF-α stimulation) and negative controls (dominant-negative IκB expression).

• Chromatin immunoprecipitation (ChIP) assays:

  • Use p65 antibodies to immunoprecipitate chromatin complexes.

  • Perform qPCR to detect enrichment of NF-κB target gene promoters.

  • Analyze how GNG12 manipulation affects p65 binding to target promoters.

• PD-L1 expression analysis:

  • Measure PD-L1 mRNA levels using qRT-PCR and protein levels using Western blotting in the context of GNG12 manipulation .

  • Experimental designs should include:

    • GNG12 knockdown

    • GNG12 overexpression

    • Combined GNG12 and p65 knockdown to establish dependence

  Results from published studies indicate that:

  • Knocking down GNG12 down-regulates PD-L1 at both mRNA and protein levels

  • GNG12 overexpression increases PD-L1 expression

  • The effects of GNG12 on PD-L1 expression are mediated through p65/NF-κB pathway

• Co-immunoprecipitation (Co-IP) assays:

  • Use GNG12 antibodies to immunoprecipitate protein complexes.

  • Perform Western blotting to detect interaction with components of the NF-κB signaling pathway.

  • Validate interactions using reciprocal Co-IPs.

How can GNG12 phenotypic data from mouse models inform antibody selection and experimental design in translational research?

Mouse models provide valuable insights into GNG12 function and can guide antibody selection for translational research:

• Phenotypic relevance to experimental design:

  • Mouse phenotypic data indicates that Gng12 mutations affect multiple physiological parameters including circulating phosphate and calcium levels, grip strength, startle reflex, and ocular abnormalities .

  • These phenotypes suggest GNG12's involvement in diverse physiological processes, which should inform experimental design when targeting specific aspects of GNG12 function.

• Selection of appropriate antibodies for cross-species studies:

  • When designing experiments that bridge mouse models and human samples, select antibodies with validated reactivity to both species.

  • Examples include:

    • ABIN6262016 (reacts with human and mouse)

    • ABIN6243817 (reacts with human and mouse)

    • 15781-1-AP (reacts with human, mouse, and rat)

• Tissue-specific considerations:

  • Given the ocular phenotypes observed in Gng12 mutant mice (abnormal lens morphology, retina blood vessel abnormalities, anophthalmia, cataract) , special attention should be paid to antibody performance in ocular tissues.

  • For nervous system studies (suggested by the increased startle reflex phenotype) , validate antibodies specifically for neural tissue applications.

• Genetically modified mouse models:

  • The available mouse models (e.g., C57BL/6NJ-Gng12 em1(IMPC)J/Mmjax and C57BL/6NCrl-Gng12 em1(IMPC)Tcp/Cmmr) can serve as excellent specificity controls for antibody validation.

  • Wild-type tissues provide positive controls, while tissues from homozygous knockout mice offer negative controls.

• Xenograft models:

  • For cancer research applications, consider the established xenograft mouse models where GNG12 manipulation altered cancer progression .

  • These models demonstrated that GNG12 knockdown impeded xenograft growth in vivo, suggesting important points for antibody application in monitoring treatment responses.

What are common pitfalls in GNG12 western blotting experiments and how can they be resolved?

Western blotting for GNG12 presents several technical challenges due to its small size (8 kDa) and specific characteristics:

• Poor detection or weak signal:

  • Problem: GNG12's small size (8 kDa) may result in poor transfer efficiency or diffusion during electrophoresis.

  • Solution:

    • Use higher percentage gels (15-20%) for better resolution

    • Optimize transfer conditions (shorter time, lower voltage for small proteins)

    • Consider PVDF membranes with smaller pore sizes (0.2 μm instead of 0.45 μm)

    • Use wet transfer systems rather than semi-dry for small proteins

    • Try longer primary antibody incubation times (overnight at 4°C)

• Multiple bands or non-specific binding:

  • Problem: Some GNG12 antibodies may detect multiple bands.

  • Solution:

    • Increase blocking time and concentration (5% milk or BSA for 2 hours)

    • Optimize antibody dilution (test a range from 1:500 to 1:5000)

    • Include GNG12 knockdown or knockout controls to identify specific bands

    • Use antibodies targeting different epitopes to confirm results

• Inconsistent results between experiments:

  • Problem: Variation in GNG12 detection between experiments.

  • Solution:

    • Standardize lysate preparation (consistent lysis buffer, concentration)

    • Ensure complete protein denaturation (adequate boiling time in sample buffer)

    • Consider using freshly prepared samples rather than freeze-thawed lysates

    • Implement rigorous loading controls appropriate for small proteins

• Sample preparation issues:

  • Problem: G proteins may show differential extraction efficiency depending on cellular localization.

  • Solution:

    • Use lysis buffers containing appropriate detergents (RIPA or modified buffers)

    • Include protease inhibitors to prevent degradation

    • For membrane-associated forms, ensure adequate solubilization

How can discrepancies in GNG12 expression data between different detection methods be reconciled and interpreted?

Researchers often encounter discrepancies when measuring GNG12 expression using different methodologies. Here's how to approach such conflicts:

• Comparing IHC with Western blot results:

  • Potential discrepancy: IHC might show positive staining while Western blot shows weak bands.

  • Reconciliation approach:

    • Consider that IHC detects localized expression that might be diluted in whole-cell lysates

    • Verify antibody suitability for each specific application

    • Use cell fractionation in Western blotting to enrich for membrane components where G proteins often localize

    • Correlate with mRNA expression data (qRT-PCR) as a third validation method

• Discrepancies between mRNA and protein levels:

  • Potential discrepancy: High mRNA levels with low protein detection or vice versa.

  • Reconciliation approach:

    • Consider post-transcriptional regulation mechanisms

    • Evaluate protein stability and turnover rate

    • Assess translation efficiency

    • Use pulse-chase experiments to determine protein half-life

• Variability between antibodies targeting different epitopes:

  • Potential discrepancy: Different results when using antibodies targeting different regions of GNG12.

  • Reconciliation approach:

    • Consider potential post-translational modifications that might mask specific epitopes

    • Evaluate potential isoform recognition differences

    • Assess antibody validation data for each specific application

    • Use genetic approaches (siRNA/shRNA) to validate specificity

• Integration of multiple data sources:
  When analyzing GNG12 expression in cancer contexts such as PDAC, researchers should integrate:

  • Tissue microarray data

  • Western blot results

  • qRT-PCR data

  • Genomic databases like Oncomine and GEPIA

  This multi-modal approach provides a more complete picture of GNG12 expression patterns.

How does GNG12's role in cancer biology inform the development of next-generation therapeutic antibodies and diagnostics?

Recent discoveries about GNG12's role in cancer, particularly in PDAC, open new avenues for therapeutic development:

• GNG12 as a prognostic biomarker:

  • High GNG12 expression correlates with poor prognosis in PDAC patients .

  • Diagnostic applications:

    • IHC-based tissue scoring systems for patient stratification

    • Potential circulating biomarker for liquid biopsy development

    • Combination with other markers for improved prognostic accuracy

• Targeting the GNG12-NF-κB-PD-L1 axis:

  • GNG12 activates NF-κB signaling and increases PD-L1 expression .

  • Therapeutic implications:

    • Development of antibodies or small molecules targeting GNG12

    • Combination therapies with existing PD-1/PD-L1 immune checkpoint inhibitors

    • Stratification of patients for immunotherapy based on GNG12 expression

• Antibody-based therapeutic approaches:

  • Therapeutic antibodies against GNG12 could potentially:

    • Block interactions with downstream effectors

    • Facilitate internalization and degradation of GNG12

    • Deliver toxic payloads specifically to GNG12-overexpressing cancer cells

• Research directions for antibody development:

  • Humanized antibodies targeting functional domains of GNG12

  • Bispecific antibodies targeting GNG12 and components of the immune system

  • Antibody-drug conjugates for targeted delivery to GNG12-expressing cells

  • Research should focus on epitope selection that interferes with GNG12's ability to activate NF-κB signaling

What methodological innovations are needed to better investigate GNG12's interactions with other signaling pathways beyond NF-κB?

While GNG12's role in NF-κB signaling is now established , its interactions with other pathways require further investigation:

• Proximity labeling approaches:

  • BioID or APEX2 fusion proteins with GNG12 to identify proximal interacting partners

  • TurboID-based approaches for rapid labeling of transient interactions

  • Mass spectrometry analysis of labeled proteins to identify novel pathways

• Advanced microscopy techniques:

  • Super-resolution microscopy to visualize subcellular localization and co-localization with other signaling components

  • FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) to measure direct protein-protein interactions

  • Live-cell imaging with fluorescently tagged GNG12 to monitor dynamics in response to various stimuli

• Systems biology approaches:

  • Phosphoproteomics to identify changes in phosphorylation networks upon GNG12 manipulation

  • Transcriptomics combined with pathway analysis to identify affected signaling networks

  • Network analysis to place GNG12 in the context of broader signaling networks

• CRISPR-based screens:

  • CRISPR activation or interference screens to identify synthetic lethal interactions with GNG12

  • Focused screens targeting components of other G-protein coupled pathways

  • Double knockout/knockdown approaches to identify redundant or compensatory pathways

• Novel antibody applications:

  • Intrabodies for tracking and manipulating GNG12 in living cells

  • Nanobodies for targeting specific functional domains with minimal steric hindrance

  • Split-antibody complementation assays to detect protein-protein interactions

Each of these approaches will require specialized antibodies or genetic tools targeting GNG12 and should be validated using the strategies discussed in earlier sections.

These advanced methods will help expand our understanding of GNG12 beyond its currently established roles in cancer and inflammation.