RGS6 Antibody, FITC conjugated

What is RGS6 and why is it important in cellular signaling research?

RGS6 (Regulator of G protein signaling 6) is a protein that functions as a GTPase-activating protein (GAP) for G protein alpha subunits. It plays a crucial role in regulating G protein-coupled receptor (GPCR) signaling cascades by increasing the GTPase activity of G protein alpha subunits, thereby driving them into their inactive GDP-bound form . The RGS6/GNB5 dimer specifically enhances GNAO1 GTPase activity, which is important for signal transduction control. RGS6 has garnered significant research interest due to its involvement in cancer suppression, inflammatory processes, and cellular apoptosis regulation, making it a valuable target for diverse research areas including oncology, immunology, and cellular biology .

What are the recommended applications for RGS6 Antibody, FITC conjugated?

The FITC-conjugated RGS6 polyclonal antibody is primarily recommended for:

• Immunofluorescence microscopy (ICC/IF) - Typically used at dilutions of 1:100 for optimal visualization

• Flow cytometry - For detection of RGS6 expression in cell populations

• ELISA assays - Recommended at dilutions of 1:100-1:500

The antibody has been validated for reactivity with human samples, particularly in applications requiring direct fluorescent detection without secondary antibody incubation steps . The FITC conjugation provides convenient visualization in fluorescence-based applications with excitation at approximately 495 nm and emission at 519 nm, compatible with standard FITC filter sets.

What is the immunogen used to generate the RGS6 Antibody, FITC conjugated?

The polyclonal antibody is typically generated using a recombinant fragment of human RGS6 protein, specifically the region spanning amino acids 177-262 or within the region of amino acids 150-300 . This region was selected based on its antigenicity and unique sequence to RGS6, minimizing cross-reactivity with other RGS family members. The antibody is generally produced in rabbits and purified using protein G affinity chromatography before FITC conjugation, ensuring high specificity for the target protein .

How should I optimize RGS6 Antibody, FITC conjugated for immunofluorescence experiments?

For optimal immunofluorescence results with RGS6 Antibody, FITC conjugated:

• Fixation protocol: Use 4% formaldehyde or paraformaldehyde for 5-10 minutes at room temperature

• Permeabilization: Treat with 0.1-0.2% Triton X-100 for 2-5 minutes to allow antibody access to intracellular targets

• Blocking: Use 5% BSA in PBS for 30-60 minutes to reduce background staining

• Antibody dilution: Start with 1:100 dilution in blocking buffer; optimize as needed

• Incubation time: Incubate cells with antibody solution overnight at 4°C or for 1-2 hours at room temperature

• Nuclear counterstain: Use DAPI (1:1000) for 5 minutes for nuclear visualization

• Controls: Include a negative control (no primary antibody) and if possible, RGS6 knockout/knockdown cells

For multi-color immunofluorescence, ensure the FITC channel (excitation ~495 nm, emission ~519 nm) does not overlap with other fluorophores. Anti-fade mounting medium is recommended to prevent photobleaching during imaging.

What are the important considerations when using RGS6 Antibody, FITC conjugated for flow cytometry?

When using RGS6 Antibody, FITC conjugated for flow cytometry:

• Cell preparation: Single-cell suspensions must be prepared with minimal cell aggregation

• Fixation/permeabilization: Since RGS6 is predominantly intracellular, use appropriate permeabilization reagents (e.g., 0.1% saponin or commercial permeabilization kits)

• Antibody concentration: Initial recommendation of 1:100, but titration experiments should be performed (1:50, 1:100, 1:200, 1:500) to determine optimal signal-to-noise ratio

• Controls: Include:

  • Unstained cells

  • FMO (Fluorescence Minus One) control

  • Isotype control (FITC-conjugated rabbit IgG)

  • Positive control (cell line with known RGS6 expression)

  • Negative control (RGS6 knockdown/knockout cells if available)

• Compensation: If performing multi-color flow cytometry, proper compensation setup is crucial to account for FITC spectral overlap

• Data analysis: Analyze both the percentage of positive cells and mean fluorescence intensity (MFI) to quantify RGS6 expression levels

How does RGS6 Antibody, FITC conjugated perform in detecting RGS6 expression changes during apoptosis experiments?

RGS6 Antibody, FITC conjugated is particularly valuable for studying apoptosis as RGS6 has been shown to induce apoptosis via p53-independent mechanisms . When designing experiments to monitor RGS6 during apoptosis:

• Time course considerations: RGS6 expression can change during apoptosis progression, so multiple time points should be examined (typically 12h, 24h, 48h post-induction)

• Dual staining protocols: Combine RGS6 Antibody, FITC conjugated with:

  • Annexin V-APC/PI staining to correlate RGS6 expression with specific apoptotic stages

  • Active caspase-3 staining to associate RGS6 with caspase activation

• Microscopy validation: Use the same antibody for immunofluorescence to visualize RGS6 localization changes during apoptosis

• Western blot correlation: Confirm flow cytometry or immunofluorescence findings with western blot using non-conjugated RGS6 antibody

Research has demonstrated that RGS6 activates the intrinsic pathway of apoptosis by:

• Regulating Bax/Bcl-2 ratio

• Inducing mitochondrial outer membrane permeabilization (MOMP)

• Promoting cytochrome c release to the cytosol

• Activating caspase-3 and subsequent apoptotic mechanisms

For optimal results, coordinate your RGS6 antibody staining protocol with these apoptotic markers to establish temporal relationships between RGS6 expression/localization and cellular death processes.

How can RGS6 Antibody, FITC conjugated be used to investigate the role of RGS6 in cancer progression?

RGS6 has been identified as a tumor suppressor in multiple cancer types, making its detection crucial for cancer research . Advanced research applications include:

• Expression profiling in clinical samples:

  • RGS6 shows marked downregulation that correlates with cancer progression

  • Example data from breast cancer studies:

  Cancer Stage	RGS6 Expression Level (H-score)	p-value
  Normal tissue	278 ± 45	-
  Ductal carcinoma in situ	143 ± 39	<0.001
  Invasive carcinoma	67 ± 28	<0.001

• Cell survival and colony formation assays:

  • RGS6 expression inhibits colony formation by approximately 80% in breast cancer cell lines

  • Monitor changes using cell viability assays (e.g., trypan blue exclusion showing ~50% cell death within 48h of RGS6 expression)

• Metastasis and EMT research:

  • RGS6 suppresses TGF-β-induced epithelial-mesenchymal transition (EMT) in non-small cell lung cancer

  • Track changes in EMT markers (E-cadherin, vimentin, N-cadherin) in relation to RGS6 expression

• Localization studies during cancer progression:

  • Use the FITC-conjugated antibody to track changes in RGS6 subcellular localization during cancer progression

  • Co-stain with markers of different cellular compartments

For these applications, the antibody can be used in flow cytometry for quantitative assessment of RGS6 expression levels across different cancer cell populations or in immunofluorescence to visualize localization changes in tumor sections.

What are the recommended protocols for using RGS6 Antibody, FITC conjugated in inflammatory disease models?

Recent studies have implicated RGS6 in the modulation of inflammatory responses, particularly in acute lung injury models . When studying RGS6 in inflammation:

• Tissue section immunofluorescence:

  • Preparation: Fix tissue in 4% paraformaldehyde, embed in paraffin, and section at 4-5 μm thickness

  • Antigen retrieval: Heat-mediated in citrate buffer (pH 6.0)

  • Blocking: Use 5% BSA for 1 hour at room temperature

  • Antibody incubation: Apply RGS6 Antibody, FITC conjugated at 1:100-1:300 dilution overnight at 4°C

  • Co-staining: Use markers for inflammatory cells (e.g., Ly6g for neutrophils)

• Flow cytometry for inflammatory cell infiltration analysis:

  • Digest tissue samples (e.g., lung tissue) using collagenase/DNase

  • Prepare single-cell suspensions and block with Fc receptors

  • Stain with RGS6 Antibody, FITC conjugated along with specific immune cell markers

  • Key parameters to measure:

    • RGS6 expression in different immune cell populations

    • Correlation between RGS6 levels and inflammatory cytokine production

• Recommended controls for inflammation studies:

  • RGS6 knockout/knockdown models as negative controls

  • LPS-stimulated vs. unstimulated samples to assess inflammation-induced changes

Research data indicates that RGS6 knockout models show increased inflammatory response with elevated levels of IL-6, IL-1β, and MCP-1 in bronchoalveolar lavage fluid (BALF) compared to wild-type controls when challenged with inflammatory stimuli like LPS .

How can RGS6 Antibody, FITC conjugated be used to study the interaction between RGS6 and other signaling pathways?

RGS6 interacts with multiple signaling pathways beyond its canonical role in G protein regulation. Advanced studies can use the FITC-conjugated antibody to investigate:

• RGS6-SMAD signaling interactions:

  • Recent research has shown that RGS6 binds to SMAD4 to prevent complex formation between SMAD4 and SMAD2/3, thereby suppressing TGF-β signaling

  • This interaction is independent of RGS6's regulation of G-protein signaling

  • Protocol for co-localization studies:

    • Use RGS6 Antibody, FITC conjugated (green channel)

    • Counter-stain with anti-SMAD4 antibody (use a red fluorophore-conjugated secondary)

    • Perform confocal microscopy to assess co-localization

• RGS6 nuclear translocation:

  • RGS6 can affect gene expression by influencing the nuclear entry of transcription factors

  • Monitor RGS6 nuclear localization using:

    • Nuclear/cytoplasmic fractionation followed by microscopy

    • Time-course imaging after specific stimulation (e.g., TGF-β treatment)

• Proximity ligation assay (PLA) adaptation:

  • While the FITC conjugation limits traditional PLA, modified protocols can be used

  • Combine with anti-SMAD4 primary antibody and appropriate PLA probe

  • Detect RGS6-SMAD4 interaction events as fluorescent spots

This research approach has revealed that RGS6 interaction with SMAD4 results in decreased nuclear entry of phosphorylated SMAD3 and SMAD4, leading to inefficient SMAD3-mediated gene expression. This mechanism explains how RGS6 suppresses TGF-β-induced EMT and metastasis in lung cancer models .

What are common issues when using RGS6 Antibody, FITC conjugated and how can they be resolved?

For applications in fixed tissue specimens, additional considerations include:

• Optimize antigen retrieval methods (heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• For paraffin sections, ensure complete deparaffinization and rehydration

• Consider extended primary antibody incubation (overnight at 4°C) for better penetration in tissue sections

How can RGS6 Antibody, FITC conjugated be validated for specificity in research applications?

Thorough validation is essential for research accuracy. Recommended validation approaches include:

• Genetic validation:

  • Use cells with RGS6 knockout (CRISPR/Cas9) or knockdown (siRNA/shRNA)

  • Compare staining in wild-type vs. RGS6-deficient samples

  • Expected result: Reduced or absent signal in knockout/knockdown cells

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (RGS6 amino acids 177-262 or 150-300)

  • Apply to parallel samples

  • Expected result: Diminished or blocked staining

• Multiple application validation:

  • Confirm findings across different techniques (immunofluorescence, flow cytometry, western blot)

  • Correlation between techniques increases confidence in specificity

• Cross-reactivity assessment:

  • Test antibody in cells overexpressing related RGS family members

  • Expected result: Minimal cross-reactivity with other RGS proteins

• Positive control selection:

  • Known RGS6-expressing cells/tissues: HeLa cells have been validated for RGS6 expression

  • Tissues with documented expression: Colon tissue has demonstrated RGS6 expression suitable for validation

What are the best practices for quantifying RGS6 expression using FITC-conjugated antibody in different experimental systems?

Accurate quantification is critical for comparative studies. Optimal approaches include:

• Immunofluorescence quantification:

  • Image acquisition: Maintain consistent exposure settings across all samples

  • Analysis methods:

    • Mean fluorescence intensity (MFI) measurement in defined regions

    • Nuclear/cytoplasmic ratio of RGS6 signal

    • Histo-score (H-score) calculation: multiply percentage of positive cells by average intensity

  • Software recommendations: ImageJ/FIJI, CellProfiler, or specialized microscopy software

• Flow cytometry quantification:

  • Use calibration beads to standardize fluorescence intensity

  • Report both percentage of positive cells and median fluorescence intensity

  • For population studies, consider using fluorescence index: (MFI of sample)/(MFI of control)

• Tissue microarray analysis:

  • For clinical samples, use H-score system:

    • Calculate by multiplying percentage of positive cells by intensity (0-3)

    • Scale: 0-300, with higher scores indicating stronger expression

  • Published RGS6 H-scores across cancer progression stages provide benchmarks for comparison

• Dynamic expression studies:

  • For time-course experiments, normalize to appropriate housekeeping proteins

  • Consider photobleaching effects in live-cell imaging

  • For RGS6 induction/repression studies, calculate fold-change relative to baseline

How does RGS6 Antibody, FITC conjugated perform in co-localization studies with apoptotic markers?

Co-localization of RGS6 with apoptotic markers provides valuable insights into its mechanistic role in cell death. Recommended protocols:

• RGS6 and cytochrome c co-localization:

  • RGS6 has been shown to activate the intrinsic pathway of apoptosis involving cytochrome c release

  • Double immunofluorescence protocol:

    • RGS6 Antibody, FITC conjugated (1:100 dilution)

    • Anti-cytochrome c antibody with red fluorophore-conjugated secondary

    • Mitochondrial marker (e.g., MitoTracker) with far-red fluorophore

  • Analysis: Monitor temporal relationship between RGS6 expression/localization and cytochrome c release from mitochondria

• RGS6 and caspase-3 activation association:

  • Use RGS6 Antibody, FITC conjugated with SuperView™ 488 caspase-3 assay kit for live cells

  • Challenge: Both markers use green fluorescence

  • Solution: Sequential staining and imaging or parallel samples

• TUNEL assay correlation:

  • Combine RGS6 immunostaining with TUNEL assay to identify apoptotic cells

  • Use different fluorophores (e.g., TUNEL in red channel, RGS6-FITC in green)

  • Example analysis method:

    • Quantify percentage of TUNEL-positive cells expressing RGS6

    • Compare RGS6 intensity in TUNEL-positive vs. TUNEL-negative cells

Research findings indicate that cells with higher RGS6 expression show increased rates of apoptosis, with approximately 50% cell death occurring within 48 hours of RGS6 expression in breast cancer cell lines .

What are the considerations for using RGS6 Antibody, FITC conjugated to study RGS6 in cancer patient samples?

Clinical samples present unique challenges and opportunities:

• Sample preparation optimization:

  • Formalin-fixed, paraffin-embedded (FFPE) tissue sections:

    • Deparaffinization: Complete removal of paraffin is essential

    • Antigen retrieval: Optimized heat-induced epitope retrieval in citrate buffer (pH 6.0)

    • Autofluorescence quenching: Treatment with 0.1% Sudan Black B or commercial autofluorescence quenchers

• Tissue microarray (TMA) analysis:

  • High-throughput screening of RGS6 expression across multiple patient samples

  • Standardized H-score calculation for comparison across samples:

    • H-score = Σ (percentage of cells with intensity i) × (i)

    • Where i ranges from 0 (negative) to 3 (strong positive)

• Correlation with clinical parameters:

  • Research has shown that reduced RGS6 expression correlates with:

    • Advanced cancer stage

    • Decreased patient survival

    • Increased metastatic potential

  • Document clinical data including tumor stage, grade, and patient outcomes

• Multi-marker analysis:

  • Combine RGS6-FITC with markers of:

    • Proliferation (Ki-67)

    • Apoptosis (cleaved caspase-3)

    • EMT status (E-cadherin, vimentin)

  • Use sequential staining protocols to avoid fluorophore overlap

Current research indicates that RGS6 expression is significantly downregulated in breast cancer tissues compared to normal counterparts, with H-scores progressively decreasing from normal tissue (278 ± 45) to ductal carcinoma in situ (143 ± 39) to invasive carcinoma (67 ± 28) .

How can RGS6 Antibody, FITC conjugated be used in studying the role of RGS6 in regulating reactive oxygen species (ROS) production?

RGS6 has been implicated in the regulation of reactive oxygen species (ROS), an important mechanism in both cancer and inflammation. Recommended protocols:

• Combined RGS6 and ROS detection:

  • Load cells with ROS indicator (DCFDA) for 30 minutes at 37°C

  • Wash cells and fix with 2% paraformaldehyde (brief fixation to preserve DCFDA signal)

  • Permeabilize and stain with RGS6 Antibody, FITC conjugated

  • Challenge: Both DCFDA and FITC have similar emission spectra

  • Solution: Use alternative ROS indicators (e.g., DHE for superoxide, which has red fluorescence)

• Flow cytometry protocol for sequential analysis:

  • Split samples into two portions:

    • Measure ROS levels using DCFDA and flow cytometry

    • In parallel samples, assess RGS6 expression using the FITC-conjugated antibody

  • Correlate results between the two measurements

• Time-course analysis of RGS6 expression and ROS production:

  • Monitor changes in both parameters at multiple time points after stimulation

  • Example experimental design:

    • Baseline measurement

    • Short-term response (0.5, 1, 2 hours)

    • Long-term response (6, 12, 24 hours)

• RGS6 manipulation and ROS assessment:

  • Overexpress or knock down RGS6

  • Measure resulting changes in ROS production

  • Expected outcome based on research: RGS6 knockout models show increased ROS production

This approach has revealed that RGS6 plays a protective role against oxidative stress in various cell types, and its absence leads to increased ROS production, particularly in response to inflammatory stimuli .