GNA12 Antibody, FITC conjugated

What is GNA12 and what cellular functions does it regulate?

GNA12 (Guanine Nucleotide Binding Protein Alpha 12) functions as a modulator or transducer in various transmembrane signaling systems. It plays critical roles in:

• Cell migration control through TOR signaling cascade

• Regulation of biological processes associated with cancer progression

• Transmembrane signal transduction as part of G protein complexes

G proteins, including GNA12, are heterotrimeric complexes composed of three units (alpha, beta, and gamma), with the alpha chain containing the guanine nucleotide binding site responsible for GTP hydrolysis . GNA12 is primarily localized near the cell surface membrane, consistent with its role in transmitting signals from membrane-bound receptors .

What are the optimal storage conditions for GNA12 Antibody, FITC conjugated to maintain functionality?

For maximum stability and performance of FITC-conjugated GNA12 antibodies:

• Store at -20°C in buffer containing 0.02% sodium azide and 50% glycerol (pH 7.3)

• Stable for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage of small volume (20μl) sizes

• Avoid repeated freeze-thaw cycles to prevent denaturation and loss of fluorescence activity

• Protect from light exposure to prevent photobleaching of the FITC fluorophore

What are the recommended dilutions for different applications of GNA12 Antibody, FITC conjugated?

Based on validated experimental protocols, the following application-specific dilutions are recommended:

Note: It is essential to conduct titration experiments for each application and sample type to determine optimal antibody concentration and minimize background signal .

How do I troubleshoot weak signal when using GNA12 Antibody, FITC conjugated for immunofluorescence microscopy?

When encountering weak fluorescence signals in immunofluorescence experiments with FITC-conjugated GNA12 antibody, consider the following methodological improvements:

• Antigen retrieval optimization: If using fixed tissues or cells, ensure appropriate antigen retrieval (heat-induced or enzymatic) to expose epitopes masked during fixation

• Dilution adjustment: Test a more concentrated antibody dilution (1:50 instead of 1:200)

• Permeabilization enhancement: Optimize detergent concentration and incubation time to improve antibody access to intracellular GNA12

• Signal amplification: Consider implementing tyramide signal amplification (TSA) system for low-abundance targets

• Photobleaching prevention: Minimize exposure to light during processing and incorporate anti-fade mounting media

• Microscopy parameters: Adjust exposure time, gain, and detector sensitivity while avoiding autofluorescence

If GNA12 expression is expected to be low in your experimental system, consider using higher antibody concentrations or switching to a more sensitive detection method.

How can I validate the specificity of GNA12 Antibody, FITC conjugated in my experimental system?

Rigorous validation of antibody specificity is crucial for reliable results. Implement these methodological approaches:

• Positive controls: Include cell lines with confirmed GNA12 expression (e.g., HeLa or HepG2)

• Negative controls:

  • Isotype control antibody (FITC-conjugated rabbit IgG)

  • Secondary antibody-only control

  • Use of cells with GNA12 knockdown/knockout

• Epitope blocking: Pre-incubate antibody with immunogenic peptide (recombinant GNA12 AA 112-270)

• Orthogonal validation: Confirm findings using different GNA12 antibodies targeting distinct epitopes

• Western blot confirmation: Verify detection of the expected 37-39 kDa band in parallel with immunofluorescence experiments

• Subcellular localization assessment: Confirm membrane-proximal localization consistent with GNA12's known cellular distribution

What are the considerations for multiplexing GNA12 Antibody, FITC conjugated with other fluorescent markers?

When designing multiplex immunofluorescence experiments incorporating FITC-conjugated GNA12 antibody:

• Spectral compatibility: Select additional fluorophores with minimal spectral overlap with FITC (excitation ~495nm, emission ~520nm)

  Recommended Compatible Fluorophores	Excitation Peak	Emission Peak
  Cy3	~550 nm	~570 nm
  Alexa Fluor 647	~650 nm	~665 nm
  Pacific Blue	~410 nm	~455 nm

• Signal intensity balancing: Adjust individual antibody concentrations to achieve comparable signal intensities

• Sequential staining: Consider sequential rather than simultaneous antibody incubation if cross-reactivity occurs

• Appropriate controls: Include single-color controls for spectral compensation during analysis

• Antibody host species: Select primary antibodies from different host species to prevent cross-reactivity of secondary antibodies

• GNA12 colocalization studies: Prioritize membrane/subcellular markers that provide biological context for GNA12 function

How can GNA12 Antibody, FITC conjugated be employed to investigate the LPA/LPAR/GNA12 signaling pathway in cancer models?

The LPA/LPAR/GNA12 signaling pathway plays a significant role in cancer progression, particularly in ovarian cancer . Methodological approaches include:

• Proximity ligation assays: Combine GNA12-FITC antibody with LPA receptor antibodies to detect in situ protein interactions

• Flow cytometry: Quantify GNA12 expression levels across patient-derived samples with varying degrees of LPA receptor expression

• Live-cell imaging: Track dynamic changes in GNA12 localization following LPA stimulation using the direct FITC visualization

• GNA12 silencing experiments: Compare pathway activation before and after GNA12 knockdown using techniques described in :

  • Transcriptomic profiling revealed that GNA12 silencing altered expression of genes involved in:

    • PI3K/AKT signaling pathway

    • VEGF signaling

    • Chemotherapy resistance mechanisms

    • FoxO signaling

• Hub protein analysis: Investigate interactions between GNA12 and identified hub proteins in pro-tumorigenic networks:

  • AKT1, VEGFA, TGFB1, BCL2L1, STAT3 (all downregulated in GNA12-silenced cells)

Experimental design should include controls for confirming pathway activation status and appropriate inhibitors to establish causal relationships.

What methodological approaches can resolve contradictory data regarding GNA12 antibody reactivity across species?

When encountering inconsistent cross-reactivity data across species (as observed in the information sources):

• Epitope sequence analysis: Compare the conservation of the target epitope (AA 112-270) across species:

  • Perform BLAST alignment of human, mouse, and rat GNA12 sequences focusing on this region

  • Identify potential species-specific post-translational modifications

• Validation across multiple cell lines:

  • Human: HeLa, HepG2

  • Mouse: Primary tissues or established cell lines

  • Rat: Primary tissues or established cell lines

• Western blot analysis with protein loading controls:

  • Load equal protein amounts from different species

  • Confirm molecular weight differences (if any) between species

  • Compare signal intensity normalized to loading controls

• Absorption controls:

  • Pre-absorb antibody with recombinant GNA12 proteins from different species

  • Assess remaining reactivity to determine specificity

• Alternative detection methods:

  • Compare results using different anti-GNA12 antibodies with distinct epitopes

  • Consider RNA-level validation (RT-PCR) to correlate with protein detection

Different antibody lots or clones may exhibit variable cross-reactivity. Document batch information and standardize protocols when comparing studies across different laboratories.

How can GNA12 Antibody, FITC conjugated be utilized to investigate the role of GNA12 in proteasomal degradation pathways?

Recent research has revealed GNA12's involvement in suppressing proteasomal pathways , presenting an intriguing area for investigation:

• Co-localization studies with proteasome components:

  • Use GNA12-FITC antibody alongside antibodies against PSM subunits (PSMA6, PSMC5, etc.)

  • Analyze spatial relationships in response to proteasome inhibitors (e.g., bortezomib)

• Temporal dynamics analysis:

  • Track GNA12 localization changes during cell cycle progression

  • Correlate with activity of APC/C (anaphase-promoting complex/cyclosome)

• Ubiquitination pathway interaction:

  • Investigate GNA12 relationship with UBE2E1 (E2-ubiquitin conjugating enzyme)

  • Analyze impact on PRC1-mediated silencing of tumor suppressor genes

• Functional assays:

  • Compare proteasome activity in cells with normal vs. silenced GNA12 expression

  • Assess impact on programmed cell death pathways

• Therapeutic intervention assessment:

  • Monitor GNA12 expression/localization changes following treatment with:

    • Proteasome inhibitors

    • Ubiquitin-proteasome system modulators

    • Cell cycle checkpoint inhibitors

Research indicates GNA12 silencing upregulates components of the proteasomal machinery including PSMA6, PSMC5, ANAPC1, and UBE2E1 , suggesting a regulatory role worth exploring through these methodological approaches.

How should flow cytometry data using GNA12 Antibody, FITC conjugated be analyzed and interpreted?

When analyzing flow cytometry data generated with FITC-conjugated GNA12 antibody:

• Gating strategy optimization:

  • Gate on viable cells using appropriate viability dye

  • Remove doublets using FSC-H vs. FSC-A

  • Include unstained, isotype, and FMO (fluorescence minus one) controls

• Signal intensity quantification:

  • Report median fluorescence intensity (MFI) rather than mean when distribution is non-Gaussian

  • Calculate signal-to-noise ratio compared to controls

• Population heterogeneity analysis:

  • Determine if GNA12 expression is uniform or reveals distinct subpopulations

  • Correlate with functional parameters or other markers

• Intracellular vs. surface staining interpretation:

  • GNA12 is predominantly membrane-associated but may have intracellular pools

  • Permeabilization protocol efficiency affects intracellular detection

• Compensation considerations:

  • FITC spillover into PE channel requires appropriate compensation

  • Include single-color controls for each fluorophore

• Data normalization for comparison:

  • Express results as fold change over control or reference population

  • Consider ratios to housekeeping proteins for relative quantification

What are the critical considerations when designing experiments to investigate GNA12's role in cancer signaling pathways?

When exploring GNA12's involvement in cancer signaling using FITC-conjugated antibodies:

• Cell model selection:

  • Use cell lines with confirmed GNA12 expression (HeLa, HepG2)

  • Consider patient-derived models for clinical relevance

  • Include both GNA12-high and GNA12-low expressing systems

• Pathway activation triggers:

  • LPA (lysophosphatidic acid) stimulation to activate LPAR/GNA12 pathway

  • Serum starvation followed by reintroduction to observe dynamic changes

• Downstream signaling assessment:

  • Monitor key hub nodes: AKT1, VEGFA, TGFB1, BCL2L1, STAT3

  • Assess activation status through phosphorylation-specific antibodies

• Inhibitor controls:

  • Include G protein signaling inhibitors (e.g., pertussis toxin as negative control)

  • Use pathway-specific inhibitors (PI3K/AKT, VEGF) to establish causality

• Gene silencing approaches:

  • Design appropriate siRNA/shRNA controls

  • Rescue experiments to confirm specificity

  • CRISPR/Cas9 for complete GNA12 knockout

• Temporal considerations:

  • Acute vs. chronic effects of GNA12 modulation

  • Time-course experiments to capture signaling dynamics

Research has established that GNA12 drives ovarian cancer progression by upregulating a pro-tumorigenic network while downregulating growth-suppressive networks , highlighting the importance of examining both positive and negative regulatory mechanisms.

How can inconsistencies in observed molecular weight of GNA12 be resolved in experimental data?

When addressing discrepancies in the observed molecular weight of GNA12:

• Expected vs. observed molecular weight:

  • GNA12 theoretical weight: ~44 kDa

  • Observed in Western blot: 37-39 kDa

• Sources of variation:

  • Post-translational modifications (phosphorylation, ubiquitination)

  • Proteolytic processing or alternative splicing

  • SDS-PAGE running conditions (reducing vs. non-reducing)

  • Gel percentage and buffer system

• Technical validation approaches:

  • Use molecular weight markers appropriate for the expected range

  • Include positive control lysates (HeLa, HepG2)

  • Compare multiple anti-GNA12 antibodies targeting different epitopes

• Methodological considerations:

  • Sample preparation (lysis buffer, protease inhibitors)

  • Protein denaturation conditions (temperature, time)

  • Gel running conditions (voltage, time)

• Alternative confirmation methods:

  • Mass spectrometry identification of the detected band

  • Immunoprecipitation followed by Western blotting

  • Overexpression of tagged GNA12 as size reference

Careful documentation of experimental conditions and standardized protocols are essential for reproducible molecular weight determination and meaningful comparisons across studies.

How can GNA12 Antibody, FITC conjugated be utilized in studying the relationship between GNA12 and chemotherapy resistance?

Based on research linking GNA12 to therapy resistance , these methodological approaches can be employed:

• Ex vivo patient sample analysis:

  • Compare GNA12 expression levels in responsive vs. resistant tumors

  • Correlate with expression of drug resistance proteins

• Cell line models of acquired resistance:

  • Monitor GNA12 expression changes during resistance development

  • Assess reversibility upon GNA12 silencing

• Flow cytometric approaches:

  • Use GNA12-FITC antibody to sort cell populations based on expression levels

  • Evaluate chemosensitivity of GNA12-high vs. GNA12-low populations

• Co-expression analysis:

  • Correlate GNA12 with AKT1 (which is associated with paclitaxel resistance)

  • Investigate relationship with multi-drug resistance proteins

• Signaling pathway intervention:

  • Test combination of chemotherapeutics with GNA12 pathway inhibitors

  • Evaluate restoration of chemosensitivity

• Biomarker development:

  • Assess GNA12 as predictive biomarker for therapy response

  • Develop standardized quantification protocols

Research has demonstrated that GNA12 silencing affects multiple pathways associated with chemotherapy resistance , suggesting its potential as a therapeutic target to overcome resistance mechanisms.

What methodological considerations are important when using GNA12 Antibody, FITC conjugated for high-content imaging analysis?

For robust high-content imaging using FITC-conjugated GNA12 antibody:

• Sample preparation optimization:

  • Cell density considerations to prevent overlapping

  • Fixation protocol optimization to preserve epitope accessibility

  • Permeabilization conditions for intracellular access

• Image acquisition parameters:

  • Exposure time standardization to prevent photobleaching

  • Z-stack collection for 3D distribution analysis

  • Pixel resolution appropriate for subcellular localization

• Quantification approaches:

  • Integrated intensity measurement

  • Object-based analysis for subcellular structures

  • Colocalization coefficient calculation with other markers

• Data normalization strategies:

  • Cell size/shape considerations

  • Background subtraction methodology

  • Reference channel inclusion

• Advanced analytical techniques:

  • Machine learning algorithms for pattern recognition

  • Population heterogeneity assessment

  • Temporal dynamics tracking in live cell experiments

• Reproducibility considerations:

  • Technical replicate incorporation

  • Positive/negative controls on each plate

  • Regular microscope calibration verification

When analyzing GNA12 distribution, focus on membrane localization patterns consistent with its role in transmembrane signaling , and consider analysis of redistribution following receptor activation.