GNG12 Antibody, HRP conjugated

What is GNG12 and what biological functions does it have in cellular signaling?

GNG12 (Guanine nucleotide-binding protein subunit gamma-12) is a member of the G protein family that functions as a modulator or transducer in various transmembrane signaling systems. The beta and gamma chains of G proteins are required for GTPase activity, replacement of GDP by GTP, and G protein-effector interactions . GNG12 has a calculated molecular weight of 8 kDa and consists of 72 amino acids .

Recent research has revealed several critical biological functions of GNG12:

• Regulation of cell growth and casein synthesis through the mammalian target of rapamycin complex 1 (mTORC1) pathway via interaction with Ragulator

• Modulation of inflammatory signaling cascades, with evidence that GNG12 can block inflammatory responses induced by lipopolysaccharide

• Activation of nuclear factor-κB (NF-κB) signaling and promotion of programmed death-ligand 1 (PD-L1) expression in pancreatic cancer cells

• Promotion of pancreatic cancer cell growth both in vivo and in vitro

• Association with poor prognosis in pancreatic ductal adenocarcinoma (PDAC) patients when highly expressed

Additionally, the lncRNA GNG12-AS1, which is transcribed in an antisense orientation to GNG12, plays roles in breast cancer where it is coordinately downregulated with DIRAS3 and in glioma progression through the AKT/GSK-3β/β-catenin signaling pathway .

What applications is GNG12 Antibody, HRP conjugated suitable for in laboratory research?

GNG12 Antibody, HRP conjugated is primarily designed for the following applications:

The HRP (horseradish peroxidase) conjugation makes this antibody particularly useful for chemiluminescent detection systems, offering high sensitivity for detecting low abundance proteins . This conjugation eliminates the need for secondary antibodies in Western blotting protocols, simplifying workflows and potentially reducing background issues.

When using this antibody for Western blotting applications, researchers should note that the optimal dilution may be sample-dependent, and titration in each testing system is recommended to obtain optimal results . The antibody is optimized to work with chemiluminescent substrates such as Azure Radiance for maximum sensitivity .

What is the species reactivity profile of commercially available GNG12 Antibody, HRP conjugated products?

The reactivity profile of GNG12 antibodies, including HRP-conjugated versions, varies by manufacturer. Based on available data:

For HRP-conjugated versions specifically, most commercial products have been validated for human GNG12, with some showing cross-reactivity with mouse and rat samples. When selecting an antibody for your research, it's crucial to verify the specific reactivity profile from the manufacturer, especially if working with non-human models .

The species reactivity is determined by the conservation of the epitope sequence across species. Most GNG12 antibodies are raised against synthetic peptides derived from human GNG12 sequences, with some targeting specific amino acid regions (e.g., AA 25-55 or AA 2-17) .

How should GNG12 Antibody, HRP conjugated be stored to maintain optimal activity?

To maintain optimal activity, GNG12 Antibody, HRP conjugated should be stored according to these guidelines:

• Storage temperature: -20°C is recommended for long-term storage

• Buffer composition: The antibody is typically provided in PBS with additives such as:

  • 50% Glycerol

  • 0.02-0.05% sodium azide or Proclin300

  • 0.5% BSA

  • pH 7.3

• Light sensitivity: HRP-conjugated antibodies should be protected from light exposure to prevent photobleaching of the conjugate

• Stability: Generally stable for one year after shipment when stored properly

• Aliquoting: While some manufacturers indicate that "aliquoting is unnecessary for -20°C storage," dividing the antibody into small aliquots is generally recommended to avoid repeated freeze-thaw cycles for frequently used reagents

For optimal results, always follow the specific storage instructions provided by the manufacturer, as formulations may vary slightly between products.

What methodological approaches can be used to validate GNG12 Antibody, HRP conjugated specificity for critical research applications?

For rigorous research applications, validating antibody specificity is essential. The following methodological approaches are recommended for GNG12 Antibody, HRP conjugated:

Genetic validation:

• Utilize GNG12 knockdown/knockout cells: Compare signal between control and GNG12-deficient samples created using shRNA or CRISPR-Cas9 technology, as demonstrated in studies of GNG12 function in pancreatic cancer

• Overexpression validation: Compare signal between control and GNG12-overexpressing cells to confirm signal increases proportionally with expression levels

Analytical validation:

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide to demonstrate specific blocking of the signal

• Multiple antibody approach: Confirm findings using a second antibody targeting a different epitope of GNG12

• Western blot molecular weight validation: Confirm detection of a single band at the expected molecular weight of GNG12 (8 kDa)

Experimental controls:

• Positive controls: Include samples known to express GNG12 (e.g., HEK-293 cells, pancreatic cancer cell lines)

• Negative controls: Include tissues known to have low GNG12 expression

• Loading controls: Normalize protein loading using housekeeping proteins such as GAPDH

• Isotype controls: Use an irrelevant antibody of the same isotype to assess non-specific binding

Implementation of these validation approaches increases confidence in research findings and addresses the growing concern regarding antibody specificity in the scientific community.

How can GNG12 Antibody, HRP conjugated be optimized for investigating the role of GNG12 in NF-κB signaling pathways in cancer models?

Optimizing GNG12 Antibody, HRP conjugated for investigating NF-κB signaling requires a multifaceted approach:

Experimental design considerations:

• Establish appropriate cell models: Use pancreatic cancer cell lines with known GNG12 expression levels, such as PANC-1 cells, which have been used to demonstrate that GNG12 activates NF-κB signaling and increases PD-L1 expression

• Implement genetic manipulation:

  • Knockdown GNG12 using shRNA to observe decreased NF-κB activity

  • Overexpress GNG12 to observe enhanced NF-κB signaling

  • Use p65 knockdown to confirm the NF-κB-dependent effects of GNG12

• Design co-immunoprecipitation experiments:

  • Use GNG12 Antibody to immunoprecipitate GNG12 and its binding partners

  • Perform Western blot analysis for NF-κB pathway components

  • Detect interactions between GNG12 and key signaling proteins

Technical optimization:

• Signal enhancement strategies:

  • Optimize antibody dilution (typically 1:5000-1:50000 for Western blots)

  • Use high-sensitivity chemiluminescent substrates optimized for HRP detection

  • Employ signal amplification methods for detecting low-abundance interactions

• Multiplexed detection approaches:

  • Strip and reprobe membranes to detect multiple proteins on the same blot

  • Use differently labeled antibodies for simultaneous detection of GNG12 and NF-κB components

The specific finding that GNG12 knockdown down-regulates both protein and mRNA levels of PD-L1 transcriptionally in pancreatic cancer cells provides a valuable readout for assessing the functional activity of the GNG12-NF-κB axis .

What strategies can be employed to troubleshoot non-specific background or weak signal when using GNG12 Antibody, HRP conjugated in Western blotting?

When troubleshooting Western blotting with GNG12 Antibody, HRP conjugated, consider these systematic approaches:

For high background issues:

• Blocking optimization:

  • Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

  • Test different blocking agents (5% non-fat milk, 3-5% BSA, commercial blocking reagents)

  • Ensure thorough washing between steps using fresh buffers

• Antibody optimization:

  • Dilute the antibody further (start with 1:50000 dilution and titrate as needed)

  • Reduce antibody incubation time or temperature

  • Pre-absorb antibody with non-specific proteins

• Membrane and transfer considerations:

  • Ensure proper transfer of low molecular weight proteins (8 kDa) using appropriate methods

  • Use PVDF membranes instead of nitrocellulose for better protein retention and signal-to-noise ratio

  • Consider using Azure Biosystems' Radiance Q or ECL substrates for optimal detection

For weak signal issues:

• Protein loading and transfer:

  • Increase protein loading for low-abundance targets

  • Use specialized transfer conditions for small proteins like GNG12 (8 kDa)

  • Confirm transfer efficiency with reversible membrane staining

• Antibody and detection enhancement:

  • Increase antibody concentration (start with 1:5000 dilution)

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

  • Use high-sensitivity substrates designed for HRP detection

  • Increase exposure time during imaging

• Sample preparation:

  • Optimize lysis conditions to ensure complete extraction of membrane-associated G proteins

  • Add protease inhibitors to prevent degradation

  • Avoid repeated freeze-thaw cycles of samples

For persistent issues, utilizing an HRP stripping buffer to remove antibodies and reprobe membranes can help troubleshoot whether the problem lies with the primary antibody or detection system .

How can GNG12 Antibody, HRP conjugated be used to investigate the interaction between GNG12 and the mTORC1 pathway in cell growth regulation?

To investigate GNG12's interaction with the mTORC1 pathway using GNG12 Antibody, HRP conjugated, the following comprehensive methodology is recommended:

Experimental approach:

• Cell model selection:

  • Use cow mammary epithelial cells (CMECs) or similar models where GNG12's role in the mTORC1 pathway has been established

  • Include conditions with and without leucine supplementation, as research shows leucine absence decreases GNG12 expression and lysosomal localization

• Co-immunoprecipitation studies:

  • Use GNG12 Antibody for immunoprecipitation

  • Perform Western blotting with HRP-conjugated GNG12 antibody to confirm pull-down efficiency

  • Probe for mTORC1 pathway components (mTOR, Raptor, Ragulator components)

  • Validate interactions through reciprocal co-immunoprecipitation

• Activation state analysis:

  • Monitor phosphorylation of mTORC1 targets (S6K1, 4E-BP1) in response to GNG12 overexpression or knockdown

  • Use positive controls like rapamycin treatment to confirm pathway inhibition

  • Compare results between normal and GNG12-manipulated cells

Technical considerations:

• Sample preparation:

  • Use gentle lysis conditions to preserve protein-protein interactions

  • Include phosphatase inhibitors to maintain phosphorylation states

  • Fractionate cell lysates to examine cytosolic versus membrane-associated GNG12

• Western blotting optimization:

  • Use dilution ratios optimized for the specific antibody (typically 1:5000-1:50000)

  • Include appropriate molecular weight markers to identify GNG12 (8 kDa)

  • Employ appropriate controls to validate the specificity of detected interactions

Research has demonstrated that GNG12 activates the mTORC1 pathway via interaction with Ragulator, and that GNG12 overexpression can partially restore cell growth, casein synthesis, and mTORC1 signaling that is decreased in response to leucine absence . These findings provide valuable positive controls for validating experimental results.

What are the best practices for using GNG12 Antibody, HRP conjugated in studying the relationship between GNG12 and long non-coding RNA GNG12-AS1 in cancer progression?

To investigate the complex relationship between GNG12 and GNG12-AS1 in cancer, researchers should employ the following methodological approaches:

Experimental design:

• Model system selection:

  • Use breast cancer cell lines with known imprinting status of DIRAS3 (normal cell lines like HB2 and Hs27 with imprinted DIRAS3, and cancer cell lines like SUM159 and CAL51 with loss of imprinting)

  • Consider glioma cell lines, as GNG12-AS1 has been shown to affect glioma cell proliferation and migration

• Multi-omics approach:

  • Correlate protein expression of GNG12 (detected with HRP-conjugated antibody) with RNA expression of GNG12-AS1

  • Perform RNA-seq to identify all splice variants of GNG12-AS1, which has multiple splice variants

  • Use chromatin immunoprecipitation (ChIP) to analyze epigenetic regulation at the GNG12/GNG12-AS1 locus

• Functional studies:

  • Implement siRNA silencing of GNG12-AS1 to distinguish between transcriptional and post-transcriptional effects

  • Use GNG12 Antibody, HRP conjugated to monitor GNG12 protein levels after GNG12-AS1 manipulation

  • Investigate downstream pathways including AKT/GSK-3β/β-catenin signaling implicated in glioma progression

Technical considerations:

• RNA-protein correlation analysis:

  • Extract both RNA and protein from the same samples to allow direct correlation

  • Use quantitative real-time PCR with PCR Miner software for accurate GNG12-AS1 expression measurement

  • Normalize Western blot data appropriately for accurate protein quantification

• Epigenetic investigations:

  • Consider allele-specific expression analysis, as GNG12-AS1 shows allele-specific splicing in some tissues

  • Examine the role of cohesin in the regulation of GNG12-AS1 splicing

Research has shown that silencing GNG12-AS1 with siRNA complementary to its exon 1 suppresses transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding, with consequent upregulation of DIRAS3 . This approach can be combined with GNG12 protein detection to unveil the regulatory relationship between the lncRNA and its protein counterpart.

How can GNG12 Antibody, HRP conjugated be integrated into multiplexed assays to study G protein-mediated chemokine receptor signaling in immune cells?

Integrating GNG12 Antibody, HRP conjugated into multiplexed assays for studying G protein-mediated chemokine receptor signaling requires sophisticated methodological approaches:

Multiplexed detection strategies:

• Sequential immunoblotting:

  • First probe for phosphorylated signaling components using phospho-specific antibodies

  • Strip and reprobe membranes for GNG12 using HRP-conjugated antibody

  • Finally detect total protein levels of signaling components

  • Use specialized HRP stripping buffers for efficient antibody removal between probing steps

• Multi-color fluorescent Western blotting:

  • Combine GNG12 Antibody, HRP conjugated (using specific HRP substrates that produce precipitating products) with fluorescently-labeled antibodies against other targets

  • Image using systems capable of detecting both chemiluminescence and fluorescence

• Proximity-based assays:

  • Employ proximity ligation assays to detect interactions between GNG12 and other G protein subunits or chemokine receptors

  • Use HRP-conjugated GNG12 antibody with complementary primary antibodies against interaction partners

Experimental context:

• T cell migration studies:

  • Research has established that protein geranylgeranylation, which modifies γ-subunits of chemokine receptor-associated heterotrimeric small GTPases (including GNG12), is required for chemokine receptor-proximal signaling

  • Use GNG12 Antibody, HRP conjugated to monitor GNG12 expression in T cells with conditional deletion of geranylgeranyl transferase I (Pggt1b) to correlate with migration defects

  • Combine with chemokine receptor expression analysis to establish signaling relationships

• Co-immunoprecipitation strategies:

  • Precipitate chemokine receptors and probe for co-precipitated GNG12

  • Perform reverse co-immunoprecipitation using antibodies against GNG12

  • Analyze other G protein subunits in the same complexes

These approaches allow researchers to investigate how post-translational modifications of GNG12 (particularly geranylgeranylation) affect its function in chemokine receptor signaling, which is critical for T cell migration and adaptive immune responses .