GNA12 Antibody, HRP conjugated

What is the exact specificity of the GNA12 Antibody (HRP conjugated)?

The GNA12 antibody (HRP conjugated) demonstrates specificity for amino acids 112-270 of the human Guanine nucleotide-binding protein subunit alpha-12 (GNA12). This polyclonal antibody is raised in rabbits using recombinant Human Guanine nucleotide-binding protein subunit alpha-12 protein (112-270AA) as the immunogen, resulting in IgG isotype antibodies with >95% purity following Protein G purification . The antibody's specificity is particularly relevant for detecting the 44.3 kilodalton GNA12 protein, also known as Galpha 12, NNX3, RMP, or gep in human samples .

What experimental controls should be included when using GNA12 Antibody (HRP conjugated)?

For rigorous experimental design with GNA12 Antibody (HRP conjugated), implement the following controls:

• Positive Control: Use cell lines known to express GNA12, such as SKOV3 ovarian cancer cells as referenced in current literature

• Negative Control: Include either GNA12 knock-out/knock-down samples (e.g., SKOV3 cells with silenced GNA12 expression) or cell lines not expressing GNA12

• Isotype Control: Include rabbit IgG (HRP conjugated) at the same concentration as the GNA12 antibody to account for non-specific binding

• Technical Control: Perform parallel experiments with unconjugated GNA12 antibody followed by secondary HRP detection to compare signal specificity

These controls help distinguish true signals from background and validate experimental findings when investigating GNA12-related signaling pathways.

How should GNA12 Antibody (HRP conjugated) be optimized for ELISA assays?

Optimizing GNA12 Antibody (HRP conjugated) for ELISA requires systematic parameter adjustment:

Optimization Protocol:

• Antibody Titration: Test serial dilutions (1:500 to 1:10,000) to determine optimal concentration balancing signal strength and background

• Sample Preparation: For cellular samples, use lysis buffers containing protease inhibitors to preserve GNA12 protein integrity

• Blocking Optimization: Test multiple blocking agents (5% BSA, 5% non-fat milk, commercial blockers) to minimize background while preserving specific signal

• Incubation Parameters: Compare results from varying incubation times (1-24 hours) and temperatures (4°C, room temperature)

• Substrate Selection: For HRP detection, compare TMB, ABTS, and chemiluminescent substrates for optimal signal-to-noise ratio

This methodical approach leads to optimized signal detection while minimizing background interference, particularly important when studying GNA12's role in complex signaling cascades involved in oncogenic processes .

What are the recommended sample preparation methods when detecting GNA12 in cell or tissue samples?

For optimal detection of GNA12 in experimental samples, preparation methods should account for GNA12's membrane association and signaling network context:

Cell Sample Preparation:

• Harvest cells at 80-90% confluence to ensure adequate GNA12 expression

• Use membrane-compatible lysis buffers containing 1% NP-40 or Triton X-100 with protease inhibitor cocktail

• Perform lysis on ice for 30 minutes with periodic vortexing

• Clear lysates by centrifugation at 14,000×g for 15 minutes at 4°C

• Quantify protein concentration using BCA or Bradford assay prior to analysis

Tissue Sample Preparation:

• Flash-freeze tissue samples immediately after collection

• Homogenize tissues in RIPA buffer supplemented with phosphatase inhibitors (particularly important when studying GNA12 signaling pathways)

• Process tissues using mechanical disruption followed by sonication

• Clear homogenates by centrifugation at 16,000×g for 20 minutes at 4°C

These protocols preserve GNA12's native state and prevent degradation, particularly important when investigating its role in transmitting signals from plasma membrane receptors to downstream effectors in cancer progression models .

How can GNA12 Antibody (HRP conjugated) be utilized to investigate LPA-GNA12 signaling in ovarian cancer models?

To investigate LPA-GNA12 signaling pathways in ovarian cancer using HRP-conjugated GNA12 antibody:

Experimental Approach:

• LPA Stimulation: Treat ovarian cancer cells (e.g., SKOV3, Kuramochi) with lysophosphatidic acid (LPA) at varying concentrations (0.1-10 μM) and timepoints (5min-24h)

• Pathway Inhibition: Include parallel samples treated with G-protein signaling inhibitors to confirm specificity

• ELISA Detection: Utilize the HRP-conjugated GNA12 antibody to quantify changes in GNA12 protein levels or post-translational modifications

• Downstream Analysis: Compare GNA12-regulated gene expression using transcriptomic approaches similar to those described in current literature

Results Interpretation Table:

This approach provides quantitative data on GNA12's role in transmitting signals from LPA receptors to downstream oncogenic networks, advancing understanding of ovarian cancer pathophysiology .

What troubleshooting approaches can resolve non-specific binding issues with GNA12 Antibody (HRP conjugated)?

When encountering non-specific binding with HRP-conjugated GNA12 antibody, implement this systematic troubleshooting approach:

Troubleshooting Protocol:

• Blocking Optimization:

  • Test alternative blocking solutions (1-5% BSA, casein, commercial blockers)

  • Extend blocking time to 2-3 hours at room temperature

  • Add 0.1-0.3% Tween-20 to all wash and incubation buffers

• Antibody Dilution Series:

  • Prepare a broader dilution series (1:500 to 1:20,000)

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

• Sample Preparation Refinement:

  • Pre-clear samples with Protein G beads to remove components causing non-specific binding

  • Include detergent titration in sample buffers (0.05-0.3% Tween-20)

• Control Integration:

  • Run parallel wells with rabbit IgG-HRP at equivalent concentration

  • Include GNA12-depleted samples (using siRNA knockdown as described in research)

This approach systematically identifies and eliminates sources of non-specific binding, particularly important when studying GNA12 in complex samples where other G-protein subunits may cross-react with the antibody.

How does GNA12 interface with proteasomal pathways in cancer progression?

Recent transcriptomic analyses reveal a complex relationship between GNA12 and proteasomal regulation:

GNA12 appears to suppress a growth-inhibitory network involving multiple proteasome components. When GNA12 is silenced in ovarian cancer cells, research shows upregulation of proteasome components including proteasome 20S subunit (PSM) β6, PSMα6, PSM ATPase 5, ubiquitin conjugating enzyme E2 E1 (UBE2E1), PSM non-ATPase 10, and anaphase promoting complex subunit 1 (ANAPC1) . This suggests GNA12 normally suppresses these components.

Mechanistic Implications:

• Proteasomal proteolytic machinery can induce death receptor-mediated apoptosis in specific contexts

• UBE2E1 (upregulated when GNA12 is silenced) can complex with polycomb repressive complex 1 (PRC1), potentially affecting tumor suppressor silencing

• ANAPC1, also upregulated with GNA12 silencing, is part of APC/C, which regulates cell cycle arrest by marking cyclins for proteasomal degradation

These findings suggest GNA12 antibodies can be valuable tools for investigating the intersection of G-protein signaling and proteasomal regulation in cancer cells, potentially revealing new therapeutic targets at this unexplored interface.

What are the key signaling nodes to monitor when studying GNA12-mediated oncogenic networks using this antibody?

When using GNA12 antibody to investigate oncogenic networks, focus on these critical signaling nodes identified through transcriptomic and bioinformatic analyses:

Key Pro-tumorigenic Nodes Upregulated by GNA12:

• AKT1 (Protein kinase B)

• VEGFA (Vascular endothelial growth factor A)

• TGFB1 (Transforming growth factor beta 1)

• BCL2L1 (B-cell lymphoma 2 like 1)

• STAT3 (Signal transducer and activator of transcription 3)

• IGF1 (Insulin-like growth factor 1)

• GHRH (Growth hormone releasing hormone)

Growth-Suppressive Network Downregulated by GNA12:

• PSMB6 (Proteasome 20S subunit beta 6)

• PSMA6 (Proteasome 20S subunit alpha 6)

• PSMA5 (Proteasome ATPase 5)

• UBE2E1 (Ubiquitin conjugating enzyme E2 E1)

• PSMD10 (Proteasome non-ATPase 10)

• NDUFA4 (NDUFA4 mitochondrial complex-associated)

• NDUFB8 (NADH:ubiquinone oxidoreductase subunit B8)

• ANAPC1 (Anaphase promoting complex subunit 1)

Experimental Design Considerations:
When investigating these networks, design experiments to capture both direct GNA12 effects and downstream cascades, potentially through time-series analysis following LPA stimulation compared to GNA12 silencing conditions. Monitor cellular phenotypes associated with GO biological processes including cell adhesion, proliferation, and motility alongside molecular changes in these key nodes .

How does the specificity of GNA12 Antibody (HRP conjugated) targeting AA 112-270 compare with antibodies targeting other regions of the protein?

The GNA12 antibody targeting AA 112-270 region presents distinct characteristics compared to antibodies targeting other protein regions:

Comparative Specificity Analysis:

The specificity for AA 112-270 provides strategic advantages when studying GNA12's role in LPA/LPAR signaling as this region contains domains crucial for interaction with downstream effectors involved in oncogenic networks . When selecting antibodies for specific applications, researchers should consider these regional differences to optimize detection of relevant protein states and interactions.

What cross-reactivity should researchers be aware of when using GNA12 Antibody (HRP conjugated) across different species?

Understanding cross-reactivity profiles is essential for experimental design with GNA12 antibodies:

• Human-only Reactivity: Several GNA12 antibodies, including those targeting AA 112-270, AA 301-381, and Ser67, show specificity primarily for human samples

• Multi-species Reactivity:

  • Antibodies targeting the C-terminal region often show cross-reactivity with human, rat, and mouse samples

  • Some GNA12 antibodies demonstrate broader cross-reactivity across human, rat, mouse, dog, guinea pig, horse, rabbit, cow, zebrafish, hamster, monkey, and pig samples

• Sequence Conservation Considerations:
  When planning cross-species experiments, researchers should align GNA12 sequences from target species with the human AA 112-270 region to predict potential cross-reactivity. The G-protein alpha subunit shows considerable conservation across mammals, but validation is required before using in non-human models.

For definitive cross-species applications, preliminary validation experiments comparing human samples with target species are strongly recommended to confirm reactivity before conducting full experimental series.

How can GNA12 Antibody (HRP conjugated) be integrated into multiplexed detection systems for pathway analysis?

Integrating HRP-conjugated GNA12 antibody into multiplexed detection systems enables comprehensive pathway analysis:

Multiplexed Integration Strategies:

• Sequential Multiplex ELISA:

  • Utilize HRP substrate with precipitating chromogens that remain localized

  • After first detection, quench HRP activity with sodium azide or hydrogen peroxide

  • Proceed with second antibody detection using different HRP substrate color

  • This approach allows detection of GNA12 alongside other pathway components identified in oncogenic networks (AKT1, VEGFA, TGFB1)

• Antibody Microarray Integration:

  • Print capture antibodies for multiple pathway components in array format

  • Process samples across the array

  • Use GNA12 antibody-HRP as a detection reagent

  • Develop with chemiluminescent substrate for digital imaging

  • Useful for simultaneously analyzing GNA12 alongside its pro-tumorigenic network components

• Proximity Ligation Adaptation:

  • Combine GNA12 antibody-HRP with secondary proximity probes

  • Use proximity ligation to detect specific GNA12 protein-protein interactions

  • Particularly valuable for investigating interactions between GNA12 and components of the proteasomal pathway identified as being regulated by GNA12

These multiplexed approaches provide systems-level data on GNA12's integration within broader signaling networks, advancing understanding of its role in cancer progression beyond isolated protein detection.

What considerations are important when using GNA12 Antibody (HRP conjugated) in transcriptomic validation studies?

When using GNA12 Antibody (HRP conjugated) to validate transcriptomic findings, consider these important factors:

Validation Strategy Framework:

• Correlation Between Transcript and Protein Levels:

  • Transcriptomic studies have identified GNA12-regulated genes, but protein-level validation is crucial

  • Design ELISA-based validation of key hub genes identified in transcriptomic analyses (AKT1, VEGFA, TGFB1, BCL2L1, STAT3)

  • Compare relative quantification across transcript and protein levels

• Time-Course Considerations:

  • Transcriptional changes often precede protein-level changes

  • Design time-series experiments (0-72 hours) following GNA12 modulation

  • Use HRP-conjugated antibody for protein detection at multiple timepoints to capture the temporal relationship between GNA12 levels and downstream effects

• Pathway Validation:

  • Gene Ontology (GO) analysis identified enriched biological processes including cell adhesion, proliferation, and cell motility

  • Design functional assays evaluating these processes alongside GNA12 protein detection

  • Correlate protein-level changes with phenotypic outcomes to validate pathway predictions

• Causality Determination:

  • Use GNA12 antibody in rescue experiments following GNA12 silencing

  • Reintroduce wild-type or mutant GNA12 and measure restoration of downstream pathway components

  • This approach validates direct causality in GNA12-regulated transcriptional networks