RGS19 Antibody, FITC conjugated

What is RGS19 and what are its main biological functions in cellular signaling?

RGS19 (Regulator of G-protein signaling 19), also known as GAIP or GNAI3IP, functions as a critical regulator of G-protein signaling pathways. This 25 kDa protein (216 residues in mouse) inhibits signal transduction by increasing the GTPase activity of G protein alpha subunits, thereby driving them into their inactive GDP-bound form . RGS19 preferentially binds to G-alpha subfamily 1 members with a binding affinity order of G(i)a3 > G(i)a1 > G(o)a >> G(z)a/G(i)a2 .

Research has demonstrated that RGS19 plays significant roles in:

• Wnt/β-catenin signaling: RGS19 inhibits the Wnt signaling pathway through inactivation of Gαo. It attenuates Dvl phosphorylation, β-catenin accumulation, and Wnt-responsive gene transcription .

• Cardiac development: RGS19 negatively affects heart function. Transgenic mice overexpressing RGS19 exhibit septal defects, thin-walled ventricles, and reduced expression of cardiogenesis-related genes such as BMP4 and Mef2C during embryonic development .

• Autophagy regulation: RGS19 is involved in zVAD-induced autophagy and works in conjunction with GNAI3. Knockdown of RGS19 inhibits zVAD-induced LC3 modification, a marker of autophagosome formation .

• Cell death pathways: RGS19 interacts with RIP3 and is required for zVAD-induced cell death but not TNF-induced cell death in L929 cells .

What are the optimal protocols for FITC conjugation to RGS19 antibodies?

The conjugation of FITC (Fluorescein Isothiocyanate) to RGS19 antibodies involves a specific methodology to ensure optimal labeling while preserving antibody functionality. The standard protocol includes:

• Antibody preparation: Dialyze purified monoclonal antibody against 500 ml FITC labeling buffer (pH 9.2) at 4°C with 2-3 changes over 2 days. This step removes free NH₄⁺ ions and raises the pH to 9.2, which is critical for efficient conjugation .

• Concentration determination: Measure antibody concentration based on A₂₈₀ absorbance and adjust to 1-2 mg/ml for optimal conjugation efficiency .

• Conjugation reaction: Add 20 μl of 5 mg/ml FITC in anhydrous DMSO for each milligram of antibody. Incubate for 2 hours at room temperature. It's crucial that both the dye and organic solvent are anhydrous, and the FITC/DMSO solution should be prepared immediately before use .

• Purification: Remove unbound FITC by dialysis against final dialysis buffer at 4°C with 2-3 changes over 2 days .

• F/P ratio determination: Calculate the fluorescein/protein (F/P) ratio by measuring absorbance at 280 nm and 495 nm. The optimal F/P ratio typically ranges between 3 and 8 .

Commercial RGS19 Antibodies with FITC conjugates typically have excitation/emission wavelengths of 499/515 nm and are optimally detected using a 488 nm laser line in flow cytometry or fluorescence microscopy applications .

How should researchers design experiments to study RGS19's role in Wnt signaling pathway inhibition?

To effectively investigate RGS19's role in Wnt signaling inhibition, researchers should consider the following experimental design approach:

• Cell models and experimental systems:

  • Use established models like P19 teratocarcinoma cells, which have been validated for studying RGS19's effects on Wnt signaling and cardiomyocyte differentiation .

  • Consider generating stable cell lines with RGS19 overexpression using appropriate vectors (e.g., pEGFP-N1 vector driven by the CMV promoter) .

  • For in vivo studies, RGS19 transgenic mouse models can provide valuable insights into developmental effects .

• Key experimental readouts:

  • β-catenin analysis: Measure total β-catenin and phospho-β-catenin levels via Western blot to assess Wnt pathway activation state .

  • Dvl phosphorylation: Monitor Dishevelled phosphorylation as an upstream indicator of Wnt pathway activation .

  • Gene expression analysis: Quantify Wnt-responsive genes using qPCR (e.g., BMP4, Mef2C for cardiac development) .

  • Functional assays: For cardiac studies, measure expression of markers like brain natriuretic peptide, β-MHC, cardiac troponin T (cTnT), and α-myosin heavy chain (α-MHC) .

• Validation strategies:

  • Use Wnt3a protein stimulation (100 ng/ml) as a positive control to activate the pathway .

  • Include parallel experiments with RGS19 knockdown to demonstrate bidirectional effects.

  • Perform co-immunoprecipitation studies to confirm RGS19 interaction with G proteins .

• Visualization techniques:

  • Use RGS19 Antibody, FITC conjugated for immunofluorescence to track protein localization relative to Wnt pathway components.

  • Consider dual labeling with other pathway components to assess co-localization.

• Controls:

  • Include empty vector controls for overexpression studies.

  • Use non-targeting shRNA controls for knockdown experiments.

  • Wild-type mice should be included as controls for transgenic animals .

This experimental approach will provide comprehensive insights into RGS19's inhibitory role in Wnt signaling across multiple levels of the pathway.

What are the optimal applications and dilutions for RGS19 Antibody, FITC conjugated?

RGS19 Antibody, FITC conjugated can be utilized in various research applications, each requiring specific dilutions and conditions for optimal results:

General experimental considerations:

• Sample preparation: For cellular applications, fixation with 4% paraformaldehyde followed by permeabilization with 0.2% Triton X-100 for 5 minutes is generally effective .

• Buffer composition: Most protocols use 0.01M PBS, pH 7.4, with 50% glycerol as a storage buffer .

• Counterstains: Nuclei can be effectively counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for co-localization studies .

• Controls: Include isotype controls (rabbit IgG-FITC) and cells known to be negative for RGS19 expression to confirm specificity of staining.

The optimal working dilution should be determined by the end user for each specific application and experimental system, as antibody performance can vary based on sample type, fixation methods, and detection systems used .

How does RGS19 influence autophagy and cell death pathways?

RGS19 plays a significant regulatory role in autophagy and selective cell death pathways through several interconnected mechanisms:

• RGS19 in zVAD-induced autophagy:

  • RGS19 and its partner Gα subunit of Gi3 (GNAI3) are essential components required for zVAD-induced autophagy .

  • In experimental models, knockdown of RGS19 or GNAI3 significantly blocks zVAD-induced LC3 modification (LC3-II formation), which is a critical marker of autophagosome formation .

  • This effect is similar to the inhibition observed when depleting established autophagy proteins such as RIP3, RIP1, or PI3KC3 .

• Selective regulation of cell death pathways:

  • RGS19 shows pathway-specific effects on cell death: it is required for zVAD-induced cell death but has no effect on TNF-induced necroptosis in L929 cells .

  • When RGS19 is depleted using shRNA, zVAD-induced cell death is significantly blocked, while TNF-induced cell death remains unaffected .

  • This selective involvement suggests RGS19 functions at the intersection of specific death pathways rather than as a general cell death mediator.

• Interaction with RIP3:

  • RGS19 was identified as a RIP3-interacting protein through mass spectrometry analysis of RIP3 immunoprecipitates .

  • Co-immunoprecipitation experiments confirm direct interaction between RGS19 and RIP3, as well as between RGS19 and GNAI3 .

  • This interaction network creates a signaling complex that may facilitate cross-talk between G-protein and cell death pathways.

• Mechanism of autophagy regulation:

  • RGS19 appears to be involved in both zVAD- and TNF-induced autophagy, as evidenced by its effect on LC3 modification in both conditions .

  • The autophagy induced by zVAD requires not only RIP1, RIP3, PI3KC3, and Beclin-1 but also RGS19 and GNAI3 .

  • This autophagy process is required for zVAD-induced TNF production, highlighting a connection between autophagy and inflammatory signaling.

These findings indicate that RGS19 functions as a molecular switch that selectively regulates specific autophagy-dependent cell death pathways, potentially through its ability to modulate both G-protein signaling and death receptor pathways via protein-protein interactions.

What methods can be used to validate the specificity of RGS19 Antibody, FITC conjugated?

Validating antibody specificity is crucial for reliable research results. For RGS19 Antibody, FITC conjugated, several complementary approaches should be employed:

• Western Blot Validation:

  • Compare staining patterns between:

    • Wild-type cell lysates (e.g., HEK-293T cells)

    • RGS19-transfected cell lysates (showing enhanced band intensity)

    • RGS19-knockdown cells (showing reduced or absent band)

  • Verify the expected molecular weight (25 kDa for human RGS19)

  • Examine cross-reactivity with related RGS family proteins

• Immunofluorescence Controls:

  • Positive controls: Cells/tissues known to express RGS19

  • Negative controls:

    • Primary antibody omission

    • Isotype control (rabbit IgG-FITC)

    • RGS19-knockdown cells using validated shRNAs

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide before staining

• Genetic Validation:

  • Compare staining in:

    • Wild-type models

    • RGS19 overexpression models

    • RGS19 knockout/knockdown models

  • Correlation with mRNA expression (qPCR)

• Flow Cytometry Validation:

  • Confirm signal reduction after RGS19 knockdown

  • Compare staining profile with other validated RGS19 antibodies

  • Use fluorescence-minus-one (FMO) controls

• Fluorescence Properties Assessment:

  • Verify expected excitation/emission profile (499/515 nm)

  • Confirm detection with appropriate laser (488 nm)

  • Check for photobleaching characteristics

• Cross-Reactivity Testing:

  • Test reactivity across species (human, mouse) to confirm expected pattern

  • Evaluate potential cross-reactivity with other RGS family members

A robust validation strategy would include at least 3-4 of these approaches to ensure the observed signals truly represent RGS19 protein rather than non-specific binding or artifacts.

What are the critical experimental controls for studying RGS19's role in cardiac development?

• Genetic Model Controls:

  • Wild-type controls: C57BL/6 mice should be used as controls when studying RGS19 transgenic (RGS19 TG) mice .

  • Vector controls: For cell culture studies, cells transfected with empty vectors should be used alongside RGS19-expressing constructs.

  • Dose-dependent expression: Multiple transgenic lines with different RGS19 expression levels should be examined to establish dose-response relationships.

• Developmental Stage Controls:

  • Temporal controls: Analyze cardiac development at multiple embryonic stages (E9.5, E12.5, E15.5, E18.5) and postnatal time points to capture dynamic changes.

  • Spatial controls: Examine both cardiac and non-cardiac tissues to confirm tissue-specific effects.

• Molecular Pathway Controls:

  • Positive pathway controls: Include Wnt3a protein stimulation (100 ng/ml) to activate Wnt signaling as a positive control .

  • Pathway inhibitor controls: Use established Wnt pathway inhibitors alongside RGS19 to confirm mechanism.

  • Upstream/downstream marker analysis: Measure multiple components (e.g., β-catenin, phospho-β-catenin, phospho-AKT) to validate pathway effects .

• Functional Controls:

  • Cardiac marker panel: Analyze multiple cardiac markers (BMP4, Mef2C, brain natriuretic peptide, β-MHC, cardiac troponin T, α-MHC) to comprehensively assess cardiac phenotypes .

  • Electrocardiogram normalization: Include age and sex-matched controls for ECG analysis to account for normal variations.

• Technical Controls for RGS19 Antibody, FITC Conjugated:

  • Autofluorescence control: Unstained samples to establish background fluorescence levels.

  • Isotype control: Rabbit IgG-FITC at equivalent concentration to assess non-specific binding.

  • Secondary antibody control: When using indirect immunofluorescence methods.

• Cell Differentiation Controls:

  • Differentiation markers: Monitor expression of differentiation-related genes (MyoD1, Myogenin) to distinguish heart development effects from general differentiation effects .

  • Time course controls: Analyze samples at different differentiation stages (early, mid, late) to capture dynamic changes.

Implementation of these systematic controls ensures that observed phenotypes can be confidently attributed to RGS19's specific effects on cardiac development rather than to experimental artifacts or generalized developmental disruptions.

How can RGS19 Antibody, FITC conjugated be optimized for co-localization studies with other cellular markers?

Optimizing RGS19 Antibody, FITC conjugated for co-localization studies requires careful consideration of several technical aspects:

• Fluorophore Selection and Spectral Separation:

  • FITC has excitation/emission peaks at 499/515 nm , so select complementary fluorophores with minimal spectral overlap:

    • For dual labeling: Use far-red fluorophores (e.g., Alexa Fluor 647)

    • For triple labeling: Add a red fluorophore (e.g., Texas Red with emission at ~615 nm)

  • Consider using spectral unmixing algorithms for fluorophores with partial overlap

• Sequential Immunostaining Protocol:

  • Step 1: Apply RGS19 Antibody, FITC conjugated at 1:10-50 dilution

  • Step 2: Block with 5% normal serum from the species of the second primary antibody

  • Step 3: Apply unconjugated second primary antibody

  • Step 4: Use appropriate fluorophore-conjugated secondary antibody

  • Step 5: Counterstain nuclei with DAPI

• Sample Preparation Optimization:

  • Fixation: 4% paraformaldehyde provides good epitope preservation and morphology

  • Permeabilization: 0.2% Triton X-100 for 5 minutes allows antibody access to intracellular targets

  • Antigen retrieval: May be necessary for certain co-markers but must be compatible with RGS19 epitope

• Controls for Co-localization Studies:

  • Single-stained controls: Samples stained with each antibody alone to confirm signal specificity

  • Fluorescence minus one (FMO): Include all fluorophores except one to establish background

  • Non-expressed target control: Use cell lines or tissues lacking RGS19 expression

  • Peptide competition: Pre-incubate RGS19 Antibody, FITC with immunizing peptide

• Image Acquisition Parameters:

  • Sequential scanning: Capture each fluorophore separately to prevent bleed-through

  • Pinhole settings: Use identical settings (1 Airy unit) for all channels

  • Dynamic range optimization: Adjust laser power and detector gain to avoid saturation

  • Z-stack acquisition: Capture multiple focal planes for 3D co-localization analysis

• Quantitative Co-localization Analysis:

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Use intensity correlation analysis (ICA) for more detailed assessment

  • Employ object-based approaches for discrete structures

• Biological Targets of Interest for Co-localization:

  • G protein subunits: GNAI3 (RGS19 binding partner)

  • RIP3: Confirmed RGS19 interaction partner in cell death pathways

  • Wnt pathway components: β-catenin to visualize pathway inhibition

  • Autophagy markers: LC3 to study autophagy regulation

By carefully optimizing these parameters, researchers can generate reliable co-localization data to better understand RGS19's spatial relationships with interaction partners and functional targets.

What are the technical considerations for using RGS19 Antibody, FITC conjugated in flow cytometry?

Successfully employing RGS19 Antibody, FITC conjugated in flow cytometry requires attention to several technical aspects:

• Sample Preparation Protocols:

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

  • Permeabilization: Since RGS19 is primarily intracellular, use 0.1-0.2% Triton X-100 or saponin-based permeabilization buffer

  • Blocking: Include 5% BSA or 10% normal serum to reduce non-specific binding

  • Cell concentration: Maintain 1 × 10⁶ cells/100 μl for optimal staining

• Antibody Titration and Dilution:

  • Recommended starting dilution: 1:100-1:1000 for flow cytometry applications

  • Titration experiment: Test 2-fold serial dilutions (1:50, 1:100, 1:200, 1:400, 1:800) to determine optimal signal-to-noise ratio

  • Volume and incubation: Typically use 100 μl of diluted antibody per 10⁶ cells and incubate for 30-60 minutes at 4°C

• Critical Controls:

  • Unstained cells: To establish autofluorescence baseline

  • Isotype control: Rabbit IgG-FITC at equivalent concentration to assess non-specific binding

  • FMO control: Include all antibodies in panel except RGS19-FITC

  • Positive control: Cell line with known RGS19 expression

  • Negative control: RGS19 knockdown cells or cell line with minimal expression

• Instrument Configuration:

  • Excitation: 488 nm laser line optimal for FITC excitation

  • Emission filter: 530/30 nm bandpass filter to capture FITC emission peak (515 nm)

  • PMT voltage: Optimize to place negative population in first decade of log scale

  • Compensation: Required if multiplexing with other fluorophores, especially PE

• Data Analysis Considerations:

  • Gating strategy:

    • Forward/side scatter to identify intact cells

    • Singlet discrimination using FSC-H vs. FSC-A

    • Live/dead discrimination if appropriate

    • RGS19-FITC positive population based on negative controls

  • Data presentation: Report median fluorescence intensity (MFI) rather than percent positive for intracellular targets

• Special Considerations for RGS19:

  • Expression heterogeneity: RGS19 expression may vary within cell populations

  • Preservation after fixation: Confirm epitope stability with selected fixation method

  • Photobleaching: FITC is susceptible to photobleaching; protect samples from light exposure

  • Buffer compatibility: Avoid sodium azide in buffers if analyzing live cells

• Multiparameter Analysis:

  • For co-expression studies with RGS19, consider complementary fluorophores like PE, APC or PE-Cy7

  • When examining RGS19 in relation to G-protein signaling or autophagy pathways, include relevant markers like LC3 or Gα subunits

Following these guidelines will enable researchers to generate reliable flow cytometric data on RGS19 expression and correlate it with other cellular parameters.

What experimental approaches can be used to investigate RGS19 interaction with G protein subunits?

Investigating RGS19 interactions with G protein subunits requires a multi-faceted experimental approach:

• Co-Immunoprecipitation (Co-IP) Assays:

  • Standard approach: Express tagged versions (e.g., Myc-GNAI3 with Flag-RGS19) in 293T cells, immunoprecipitate with anti-Flag M2-beads, and detect interacting proteins by Western blotting with anti-Myc antibody .

  • Endogenous Co-IP: Immunoprecipitate native RGS19 using RGS19 antibodies and probe for associated G protein subunits.

  • Reverse Co-IP: Immunoprecipitate G protein subunits and detect associated RGS19.

  • Controls: Include IgG control immunoprecipitations and lysates from cells not expressing one partner.

• Proximity Ligation Assay (PLA):

  • Use RGS19 Antibody and antibodies against specific G protein subunits.

  • PLA signal will only be generated when proteins are within 40 nm of each other.

  • Quantify interaction signals per cell to assess interaction frequency.

• FRET/BRET Analysis:

  • FRET pairs: Create RGS19-CFP and Gα-YFP fusion proteins to detect energy transfer when proteins interact.

  • BRET approach: Use RGS19-Rluc and Gα-GFP fusion proteins.

  • Monitor real-time interactions in living cells under various stimulation conditions.

• GST Pull-down Assays:

  • Express GST-RGS19 fusion protein in bacteria.

  • Incubate with cell lysates containing G protein subunits or with purified G proteins.

  • Analyze pulled-down proteins by Western blot.

  • Include GDP/GTP loading conditions to assess nucleotide-dependency of interactions.

• Surface Plasmon Resonance (SPR):

  • Immobilize purified RGS19 on sensor chip.

  • Flow purified G protein subunits across the surface.

  • Measure binding kinetics (kon, koff) and affinity (KD).

  • Compare binding parameters across different G protein subunits (G(i)a3, G(i)a1, G(o)a, G(z)a/G(i)a2) .

• Functional Assays:

  • GTPase activity assays: Measure RGS19-stimulated GTP hydrolysis by G proteins.

  • Signaling readouts: Assess downstream signaling (e.g., cAMP levels, Ca²⁺ flux) in the presence/absence of RGS19.

  • Mutagenesis: Generate RGS19 mutants to identify critical residues for G protein interaction.

• Microscopy-Based Approaches:

  • Immunofluorescence co-localization: Use RGS19 Antibody, FITC conjugated with differently labeled G protein antibodies.

  • BiFC analysis: Split fluorescent protein complementation when RGS19 and G proteins interact.

  • Single-molecule imaging: Track individual molecules to assess interaction dynamics.

• Protein-fragment complementation assays:

  • Split-luciferase complementation assay

  • Split-ubiquitin yeast two-hybrid system

These complementary approaches provide a comprehensive picture of RGS19-G protein interactions, from biochemical confirmation to dynamics in living cells, with special attention to the preferential binding hierarchy observed for RGS19 with G(i)a3 > G(i)a1 > G(o)a >> G(z)a/G(i)a2 .

How does RGS19 overexpression impact cardiac development and what techniques can detect these changes?

RGS19 overexpression significantly impacts cardiac development through multiple mechanisms that can be detected using specialized techniques:

• Morphological and Structural Changes:

  • Observed phenotypes in RGS19 TG mice:

    • Septal defects during embryonic development

    • Thin-walled ventricles

    • Abnormal ventricle repolarization

  • Detection techniques:

    • Histological analysis: Hematoxylin and eosin (H&E) staining of cardiac muscle sections from normal and RGS19 TG mice hearts

    • Ultrasound imaging: Echocardiography for structural and functional assessment

    • MRI: For high-resolution 3D cardiac imaging

    • Optical projection tomography: For embryonic heart development visualization

• Molecular and Gene Expression Changes:

  • Altered gene expression:

    • Reduced expression of cardiogenesis-related genes BMP4 and Mef2C

    • Increased expression of heart failure markers: brain natriuretic peptide and β-MHC

    • Decreased expression of cardiac contractile proteins: cardiac troponin T (cTnT) and α-myosin heavy chain (α-MHC)

  • Detection techniques:

    • RT-qPCR: For quantifying mRNA expression changes

    • Western blotting: For protein level analysis of cardiac markers

    • Immunohistochemistry/Immunofluorescence: Using RGS19 Antibody, FITC conjugated along with antibodies against cardiac markers

    • RNA-seq: For genome-wide transcriptional profiling

    • ChIP-seq: To identify Wnt-responsive regulatory elements affected by RGS19

• Signaling Pathway Disruptions:

  • Affected pathways:

    • Inhibition of Wnt/β-catenin signaling

    • Altered G-protein signaling dynamics

  • Detection techniques:

    • Western blotting: For β-catenin, phospho-β-catenin, phospho-AKT levels

    • TCF/LEF reporter assays: To measure Wnt pathway activity

    • Co-immunoprecipitation: To assess protein-protein interactions

    • Phosphoproteomic analysis: To identify altered signaling networks

• Functional Cardiac Abnormalities:

  • Functional changes:

    • Abnormal ventricle repolarization

    • Altered contractility

    • Heart failure markers

  • Detection techniques:

    • Electrocardiogram (ECG) analysis: To detect repolarization abnormalities

    • Ex vivo heart perfusion: For functional assessment

    • Calcium imaging: To evaluate excitation-contraction coupling

    • Patch-clamp electrophysiology: For cardiomyocyte action potential analysis

• Cellular Phenotypes:

  • Cellular changes:

    • Increased cell proliferation

    • Altered cardiomyocyte differentiation

  • Detection techniques:

    • BrdU incorporation assays: For proliferation assessment

    • Immunocytochemistry: For cardiac-specific markers like cTnT and α-MHC in differentiating cells

    • Flow cytometry: Using RGS19 Antibody, FITC conjugated to quantify expression levels

    • Single-cell RNA-seq: To identify cell populations affected by RGS19 overexpression

These multiple approaches provide complementary data to comprehensively understand how RGS19 overexpression negatively impacts cardiac development and function through inhibition of the Wnt signaling pathway, which is critical for proper heart formation and cardiomyocyte differentiation.

What are the troubleshooting strategies for non-specific binding when using RGS19 Antibody, FITC conjugated?

When encountering non-specific binding with RGS19 Antibody, FITC conjugated, researchers can implement these systematic troubleshooting strategies:

• Optimization of Blocking Conditions:

  • Problem: Insufficient blocking leading to high background

  • Solutions:

    • Increase blocking agent concentration (try 5-10% BSA or normal serum)

    • Extend blocking time from 30 minutes to 1-2 hours

    • Use commercial blocking buffers specifically designed for immunofluorescence

    • Add 0.1-0.3% Triton X-100 to blocking buffer to reduce hydrophobic interactions

• Antibody Dilution Optimization:

  • Problem: Too concentrated antibody causing non-specific binding

  • Solutions:

    • Perform systematic titration (1:10, 1:50, 1:100, 1:500)

    • Compare signal-to-noise ratio across dilutions

    • For ELISA applications, start with 1:1000 dilution

    • For Western blot applications, optimize from 1:50-100 recommended range

• Sample Preparation Refinement:

  • Problem: Improper fixation/permeabilization affecting epitope accessibility

  • Solutions:

    • Test different fixatives (4% PFA, methanol, acetone)

    • Adjust permeabilization conditions (0.1-0.5% Triton X-100 for 5-15 minutes)

    • Try gentler permeabilization with 0.1% saponin for membrane proteins

    • Consider antigen retrieval methods if appropriate

• Buffer Optimization:

  • Problem: Salt or pH conditions promoting non-specific interactions

  • Solutions:

    • Increase wash buffer stringency (add 0.1-0.5% Tween-20)

    • Include 150-500 mM NaCl in wash buffers

    • Use TBS instead of PBS for phospho-specific applications

    • Adjust antibody diluent pH (typically 7.2-7.6)

• Pre-adsorption Techniques:

  • Problem: Cross-reactivity with similar epitopes

  • Solutions:

    • Pre-incubate antibody with excess target peptide to confirm specificity

    • Pre-adsorb on tissues/cells lacking RGS19 expression

    • Use lysates from RGS19 knockout/knockdown cells for pre-adsorption

• Additional Control Experiments:

  • Problem: Difficult to distinguish specific from non-specific signals

  • Solutions:

    • Include isotype control (rabbit IgG-FITC) at same concentration

    • Test staining on RGS19 knockdown/knockout cells or tissues

    • Compare staining pattern with non-conjugated RGS19 antibody

    • Perform peptide competition assay

• Fluorescence-Specific Issues:

  • Problem: FITC-related background or artifacts

  • Solutions:

    • Check for sample autofluorescence in unstained controls

    • Minimize exposure to light during all procedures (FITC is photosensitive)

    • Include photobleaching step before imaging if autofluorescence is high

    • Consider switching to more stable fluorophores (Alexa Fluor 488)

    • Store antibody properly at -20°C protected from light

• Application-Specific Adjustments:

  • Flow cytometry: Optimize compensation settings if multiple fluorophores are used

  • Microscopy: Adjust acquisition settings (exposure, gain) to minimize background

  • ELISA: Consider specialized blocking buffers to reduce plate binding

By systematically implementing these strategies, researchers can significantly improve specificity and reduce background when using RGS19 Antibody, FITC conjugated, leading to more reliable and interpretable experimental results.

How can researchers optimize RGS19 Antibody, FITC conjugated for live cell imaging applications?

Optimizing RGS19 Antibody, FITC conjugated for live cell imaging requires special considerations to maintain cell viability while achieving specific labeling:

• Antibody Delivery Methods:

  • Microinjection: Direct introduction of diluted antibody (1:50-1:100) into individual cells

  • Cell-penetrating peptides: Conjugate cell-penetrating peptides to facilitate antibody uptake

  • Protein transfection reagents: Commercial reagents like Chariot™ or PULSin™ designed for antibody delivery

  • Electroporation: Transient membrane permeabilization allowing antibody entry

  • Streptolysin O permeabilization: Reversible pore formation for antibody introduction

• Buffer and Media Optimization:

  • Imaging media composition:

    • Use phenol red-free media to reduce background fluorescence

    • Supplement with 10-25 mM HEPES (pH 7.4) for pH stability without CO₂

    • Include antioxidants (glutathione, vitamin C) to reduce phototoxicity

  • Antibody diluent:

    • Use 0.01 M PBS, pH 7.4 with reduced (25%) glycerol content compared to storage buffer

    • Ensure sodium azide is removed from preparation (toxic to live cells)

• Phototoxicity Management:

  • Illumination strategies:

    • Use reduced laser power or lamp intensity (30-50% of fixed sample settings)

    • Employ pulsed illumination rather than continuous exposure

    • Utilize intelligent acquisition software with minimal illumination algorithms

  • FITC considerations:

    • Be aware of FITC's relatively high photobleaching rate

    • Consider adding Trolox (vitamin E analog) as an anti-fading agent

    • Use oxygen scavengers (glucose oxidase/catalase system) to reduce phototoxicity

• Temperature and Environmental Control:

  • Maintain stable temperature (37°C) using stage or chamber incubators

  • Provide humidified atmosphere with appropriate CO₂ levels (5%)

  • Minimize exposure to ambient light during preparation and imaging

• Optical Considerations:

  • Objective selection:

    • Use high NA water-immersion objectives for better signal collection

    • Consider long working distance objectives for thick specimens

  • Acquisition settings:

    • Employ faster scanning speeds to reduce exposure times

    • Use resonant scanners for high-speed imaging

    • Optimize pinhole setting (1-1.5 Airy units) for best signal-to-noise ratio

• Validation and Controls:

  • Live/dead markers: Include viability dyes to confirm cell health during imaging

  • RGS19-fluorescent protein fusion: Compare antibody labeling pattern with RGS19-GFP fusion

  • Fixed cell comparison: Validate live cell patterns with fixed cell RGS19 distribution

  • Functionality assays: Confirm that antibody binding doesn't disrupt RGS19 function

• RGS19-Specific Considerations:

  • Focus on regions where RGS19 is more accessible (cytoplasmic vs. membrane-associated pools)

  • Monitor potential internalization or trafficking of antibody-bound RGS19

  • Consider the dynamic nature of RGS19 interactions with G proteins and impact of antibody binding

• Image Analysis Optimization:

  • Implement deconvolution algorithms to improve signal-to-noise ratio

  • Use adaptive thresholding for accurate RGS19 detection

  • Apply bleach correction algorithms for time-lapse studies

By implementing these strategies, researchers can effectively balance the challenges of maintaining cell viability while achieving specific labeling of RGS19 in live cell imaging applications, enabling dynamic studies of this important regulator of G-protein signaling.

What is the recommended protocol for studying RGS19's effect on zVAD-induced autophagy using FITC-conjugated antibodies?

The following comprehensive protocol is designed to investigate RGS19's role in zVAD-induced autophagy using RGS19 Antibody, FITC conjugated alongside other key markers:

Materials Required:

• Cell culture: L929 cells (established model for zVAD-induced autophagy)

• Treatments:

  • zVAD-fmk (20-50 μM)

  • TNF-α (10 ng/ml) for comparison

• Antibodies:

  • RGS19 Antibody, FITC conjugated

  • Anti-LC3 antibody (autophagy marker)

  • Anti-GNAI3 antibody (RGS19 partner)

• RNAi reagents:

  • shRNAs targeting RGS19, GNAI3, RIP1, RIP3, and PI3KC3

  • Non-targeting shRNA control

Cell Preparation and Treatment

• Culture L929 cells in DMEM supplemented with 10% FBS at 37°C, 5% CO₂.

• Seed cells at 5 × 10⁵ cells/well in 6-well plates for protein analysis or 2 × 10⁴ cells/well in 8-well chamber slides for microscopy.

• For knockdown studies:

  • Transduce cells with lentivirus expressing shRNAs targeting RGS19, GNAI3, RIP1, RIP3, or PI3KC3 .

  • Select stable knockdown cells with appropriate antibiotic.

  • Confirm knockdown efficiency by Western blot .

• Treat cells with:

  • zVAD-fmk (20-50 μM) in complete medium

  • TNF-α (10 ng/ml) as comparison

  • Vehicle control (DMSO at equivalent concentration)

• Incubate for 6-24 hours based on experimental endpoint.

Autophagy Detection by Western Blot

• Harvest cells and prepare lysates in RIPA buffer with protease/phosphatase inhibitors.

• Separate proteins on 15% SDS-PAGE to resolve LC3-I and LC3-II bands.

• Transfer to PVDF membrane and block with 5% non-fat milk.

• Probe with:

  • Anti-LC3 antibody (1:1000) to detect LC3-I to LC3-II conversion

  • Anti-RGS19 antibody (1:1000)

  • Anti-GNAI3 antibody (1:1000)

  • Anti-β-actin antibody (1:5000) as loading control

• Visualize using appropriate secondary antibodies and ECL detection.

• Quantify LC3-II/LC3-I ratio and LC3-II/β-actin ratio .

Immunofluorescence Analysis

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.2% Triton X-100 for 5 minutes .

• Block with 5% BSA in PBS for 1 hour.

• Stain with:

  • RGS19 Antibody, FITC conjugated (1:50 dilution)

  • Anti-LC3 antibody followed by appropriate secondary antibody (different fluorophore)

• Counterstain nuclei with DAPI .

• Mount slides with anti-fade mounting medium.

• Image using confocal microscopy with appropriate filter sets.

• Quantify:

  • LC3 puncta formation (autophagosome marker)

  • Co-localization between RGS19 and LC3 using Pearson's correlation coefficient

Flow Cytometric Analysis

• Harvest cells after treatment and fix with 4% paraformaldehyde.

• Permeabilize with 0.1% saponin in PBS.

• Stain with:

  • RGS19 Antibody, FITC conjugated (1:100-1:1000)

  • Anti-LC3 antibody conjugated to a different fluorophore

• Analyze by flow cytometry using 488 nm laser for FITC excitation .

• Quantify:

  • RGS19 expression levels (MFI)

  • LC3 expression levels

  • Correlation between RGS19 and LC3 expression

Co-immunoprecipitation Analysis

• Prepare cell lysates in non-denaturing lysis buffer.

• Immunoprecipitate RGS19 using anti-RGS19 antibody and protein A/G beads.

• Analyze immunoprecipitates by Western blotting for:

  • RIP3 (confirmed RGS19 interaction partner)

  • GNAI3 (partner G protein)

  • Autophagy proteins (Beclin-1, ATG5, etc.)

• Perform reverse Co-IP with anti-RIP3 antibody to confirm interactions.

Data Analysis and Interpretation

• Compare LC3-II levels across:

  • Control vs. zVAD-treated cells

  • Non-targeting shRNA vs. RGS19/GNAI3 knockdown cells

• Analyze co-localization patterns of RGS19 with autophagy markers.

• Correlate RGS19 expression levels with autophagy induction.

• Determine whether RGS19 knockdown affects zVAD-induced but not TNF-induced cell death .

By following this protocol, researchers can comprehensively investigate RGS19's role in zVAD-induced autophagy and establish its relationship with GNAI3, RIP3, and autophagy machinery components.

What are the protocols for quantifying RGS19 expression in cardiac tissues using FITC-conjugated antibodies?

The following detailed protocols enable precise quantification of RGS19 expression in cardiac tissues using FITC-conjugated antibodies across multiple experimental platforms:

Materials Required:

• Fixed cardiac tissue (formalin-fixed paraffin-embedded or frozen)

• RGS19 Antibody, FITC conjugated

• Nuclear counterstain (DAPI)

• Antifade mounting medium

• Cardiac marker antibodies (cTnT, α-MHC)

Protocol:

• Tissue preparation:

  • For FFPE sections: Cut 7-μm sections and mount onto glass slides

  • For frozen sections: Cut 10-μm cryosections

• Deparaffinization and antigen retrieval (for FFPE):

  • Deparaffinize in xylene (2 × 10 min)

  • Rehydrate through graded ethanol series

  • Perform heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Staining procedure:

  • Block with 5% normal serum or BSA for 1 hour at room temperature

  • Incubate with RGS19 Antibody, FITC conjugated (1:10-50 dilution) overnight at 4°C

  • For dual labeling, apply primary antibody against cardiac markers (cTnT, α-MHC)

  • Apply secondary antibody with contrasting fluorophore for cardiac markers

  • Counterstain nuclei with DAPI

  • Mount with antifade medium

• Quantitative image analysis:

  • Acquire images using confocal or fluorescence microscopy

  • Analyze 5-10 random high-power fields per sample

  • Measure mean fluorescence intensity of RGS19-FITC signal

  • Normalize to cell number (DAPI-positive nuclei)

  • Compare expression between experimental groups (e.g., normal vs. RGS19 TG mice)

Materials Required:

• Fresh cardiac tissue

• Tissue dissociation kit

• RGS19 Antibody, FITC conjugated

• Viability dye

Protocol:

• Single-cell suspension preparation:

  • Mince fresh cardiac tissue into ~1 mm³ pieces

  • Digest with collagenase/dispase enzyme mix at 37°C for 30-45 minutes

  • Filter through 70 μm cell strainer

  • Centrifuge at 300 × g for 5 minutes

  • Resuspend in PBS with 2% FBS

• Staining procedure:

  • Incubate cells with viability dye for 15 minutes

  • Fix with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.1% Triton X-100 for 10 minutes

  • Block with 2% BSA for 30 minutes

  • Stain with RGS19 Antibody, FITC conjugated (1:100-1:1000) for 30 minutes at 4°C

  • For cardiomyocyte identification, include α-MHC antibody with different fluorophore

• Flow cytometric analysis:

  • Acquire data using 488 nm laser for FITC excitation

  • Gate on:

    • Viable cells (viability dye negative)

    • Single cells (FSC-H vs. FSC-A)

    • Cardiomyocytes (α-MHC positive)

  • Measure RGS19-FITC median fluorescence intensity (MFI)

  • Compare MFI between experimental groups

Materials Required:

• Cardiac tissue lysates

• Anti-RGS19 antibody (non-conjugated)

• Fluorescent secondary antibody

• Loading control antibody (β-actin)

Protocol:

• Tissue lysis and protein extraction:

  • Homogenize cardiac tissue in RIPA buffer with protease inhibitors

  • Centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Western blot procedure:

  • Separate 30 μg protein by 12% SDS-PAGE

  • Transfer to PVDF membrane

  • Block with 5% non-fat milk

  • Incubate with anti-RGS19 antibody (1:1000-10,000)

  • Apply fluorescent secondary antibody

  • Probe for β-actin as loading control

• Quantitative analysis:

  • Capture images using fluorescence scanner

  • Measure RGS19 band intensity at 25 kDa

  • Normalize to β-actin

  • Compare expression ratios between experimental groups

Materials Required:

• Cardiac tissue samples

• RNA isolation kit

• cDNA synthesis kit

• RGS19-specific primers

• Reference gene primers

Protocol:

• RNA isolation and cDNA synthesis:

  • Extract total RNA from cardiac tissue

  • Assess RNA quality (A260/280 ratio)

  • Synthesize cDNA using random hexamers

• qPCR procedure:

  • Design primers specific for RGS19

  • Perform qPCR using SYBR Green or TaqMan chemistry

  • Include reference genes (GAPDH, 18S rRNA)

  • Run technical triplicates for each sample

• Data analysis:

  • Calculate relative expression using 2^-ΔΔCt method

  • Normalize to reference genes

  • Compare expression levels between experimental groups

  • Correlate mRNA expression with protein levels from Western blot/flow cytometry

In Situ Hybridization Combined with Immunofluorescence

This advanced technique allows simultaneous detection of RGS19 mRNA and protein:

• Perform RNAscope in situ hybridization for RGS19 mRNA

• Follow with immunofluorescence using RGS19 Antibody, FITC conjugated

• Analyze co-localization of mRNA and protein signals

These complementary approaches provide comprehensive quantification of RGS19 expression at both the mRNA and protein levels in cardiac tissues, enabling researchers to fully characterize expression patterns in normal development and disease states.

How can researchers assess the impact of RGS19 on Wnt signaling inhibition using FITC-conjugated antibodies?

To comprehensively assess RGS19's impact on Wnt signaling inhibition using FITC-conjugated antibodies, researchers should implement this multi-methodological approach:

Cellular Models and Experimental Design

• Cell models:

  • P19 teratocarcinoma cells (established model for studying RGS19 effects on Wnt signaling)

  • Generate stable cell lines:

    • RGS19-overexpressing cells

    • Vector control cells

    • RGS19 knockdown cells (shRNA)

  • Primary cardiomyocytes or cardiac progenitor cells

• Treatment conditions:

  • Wnt3a protein stimulation (100 ng/ml for 24h) as pathway activator

  • GSK3β inhibitors (e.g., CHIR99021) as alternative pathway activator

  • Various timepoints (2h, 6h, 24h) to capture dynamics

Immunofluorescence Analysis of Wnt Pathway Components

• Sample preparation:

  • Culture cells on glass coverslips

  • Fix with 4% paraformaldehyde for 15 minutes

  • Permeabilize with 0.2% Triton X-100 for 5 minutes

• Dual immunofluorescence staining:

  • RGS19 Antibody, FITC conjugated (1:10-50)

  • Anti-β-catenin antibody with contrasting fluorophore (e.g., Alexa 594)

  • DAPI nuclear counterstain

• Analysis parameters:

  • Nuclear vs. cytoplasmic β-catenin localization (key indicator of pathway activation)

  • Co-localization between RGS19 and β-catenin (Pearson's coefficient)

  • Quantify nuclear β-catenin intensity in:

    • RGS19-high vs. RGS19-low cells

    • Wnt3a-stimulated vs. unstimulated conditions

Flow Cytometric Analysis for Pathway Components

• Cell preparation:

  • Harvest cells after treatments

  • Fix and permeabilize using standard protocols

• Staining strategy:

  • RGS19 Antibody, FITC conjugated (1:100-1:1000)

  • Anti-β-catenin antibody with different fluorophore

  • Anti-phospho-β-catenin antibody with third fluorophore

• Analysis approach:

  • Measure correlation between RGS19 expression and β-catenin levels

  • Quantify phospho-β-catenin/total β-catenin ratio as function of RGS19 expression

  • Compare median fluorescence intensity across treatment conditions

Biochemical Pathway Analysis

• Western blot analysis:

  • Prepare lysates from control and RGS19-overexpressing cells

  • Analyze key Wnt pathway components:

    • β-catenin and phospho-β-catenin levels

    • Dvl phosphorylation

    • Phospho-AKT levels

    • GSK3β phosphorylation

  • Quantify protein expression normalized to loading controls

• Cellular fractionation:

  • Separate nuclear and cytoplasmic fractions

  • Quantify β-catenin distribution between compartments

  • Correlate with RGS19 expression levels

Functional Reporter Assays

• TOPFlash reporter assay:

  • Transfect cells with TCF/LEF luciferase reporter

  • Co-transfect with RGS19 expression vector or shRNA

  • Measure luciferase activity after Wnt3a stimulation

  • Correlate reporter activity with RGS19 expression

• Target gene expression analysis:

  • Quantify Wnt target genes in control vs. RGS19-expressing cells:

    • Measure BMP4 and Mef2C expression by qPCR

    • Analyze Axin2, c-Myc, and Cyclin D1 expression

  • Perform rescue experiments with constitutively active β-catenin

RGS19 and G-protein Interaction Analysis

• Co-immunoprecipitation:

  • Immunoprecipitate RGS19 using anti-RGS19 antibody

  • Probe for Gαo (RGS19 target in Wnt signaling)

  • Analyze β-catenin and Dvl presence in immunoprecipitates

• Proximity ligation assay:

  • Detect endogenous RGS19-Gαo interactions

  • Quantify interaction signals with/without Wnt stimulation

  • Correlate with β-catenin nuclear localization

Differentiation Assays in P19 Cells

• Cardiomyocyte differentiation protocol:

  • Culture P19 cells in bacterial grade dishes with 1% DMSO for 4 days

  • Transfer aggregates to tissue culture dishes

  • Monitor spontaneous beating (starting day 9-10)

• Immunofluorescence analysis:

  • Stain for cardiac markers (cTnT, α-MHC) and RGS19-FITC

  • Quantify differentiation efficiency

  • Correlate RGS19 expression with differentiation markers

• Gene expression profiling:

  • Analyze expression of cardiac markers:

    • MyoD1, Myogenin (early markers)

    • α-MHC, cTnT (late markers)

  • Correlate with RGS19 expression levels and Wnt pathway activity

In Vivo Validation Using RGS19 Transgenic Mice

• Embryonic analysis:

  • Compare Wnt pathway activity in wild-type vs. RGS19 TG mice hearts

  • Perform immunohistochemistry for β-catenin localization

  • Analyze cardiac developmental defects (septal defects, thin-walled ventricles)

• Molecular profiling:

  • Quantify β-catenin, phospho-AKT levels by Western blot

  • Measure expression of Wnt target genes in heart tissue

  • Correlate with cardiac abnormalities