GPER1 Antibody, FITC conjugated

What is GPER1 and why is it important in research?

GPER1 (G Protein-Coupled Estrogen Receptor 1) is a membrane-bound estrogen receptor that mediates rapid cellular responses to estrogen independent of classical nuclear estrogen receptors. GPER1 functions through G-protein signaling pathways, triggering the release of intracellular calcium and activating Src-like kinases required for EGFR transactivation . The receptor is significant in research because it coordinates critical cellular processes including the release of EGF-like polypeptides and formation of provisional fibronectin matrix, which facilitate cellular survival in both normal tissues and potentially in cancer cells . Furthermore, GPER1 activation has been shown to protect against oxidative stress-induced cell death in cardiomyoblasts and influence mitochondrial integrity, making it relevant for cardiovascular research .

How does FITC conjugation enhance GPER1 antibody utility in research?

FITC (Fluorescein Isothiocyanate) conjugation provides direct fluorescent labeling of the GPER1 antibody, eliminating the need for secondary antibody incubation steps in immunofluorescence and flow cytometry applications . This conjugation allows for direct visualization of GPER1 expression with green fluorescence (excitation ~495 nm, emission ~519 nm), which streamlines experimental protocols and enables multiplexing with other fluorophores that have different spectral properties. The direct conjugation also reduces background signal and non-specific binding issues that can occur with two-step detection systems, thereby improving signal-to-noise ratios in imaging and cytometry experiments.

What are the basic principles for validating GPER1 antibody specificity?

Validating GPER1 antibody specificity requires multiple complementary approaches:

• Positive and negative control samples: Use cell lines with known GPER1 expression levels. MCF-7 breast cancer cells express GPER1 and can serve as positive controls , while cells with GPER1 knockdown or tissues from GPER1 knockout models provide essential negative controls.

• Blocking peptide controls: Pre-incubation of the antibody with a specific blocking peptide should abolish positive staining in immunofluorescence or flow cytometry.

• Multiple detection methods: Confirm findings using alternative techniques such as Western blotting, qRT-PCR, or immunohistochemistry with different antibody clones.

• Pharmacological validation: Stimulation with GPER1-specific agonists like G1 should produce expected biological responses in positive-staining cells but not in cells where staining is absent .

• Subcellular localization patterns: GPER1 shows specific localization patterns that should be consistent with published literature, including plasma membrane and endoplasmic reticulum distribution.

How should samples be prepared for optimal GPER1 detection using FITC-conjugated antibodies?

Sample preparation for optimal GPER1 detection requires careful attention to preservation of both antigen integrity and native cellular architecture:

• Fixation protocol: For cell cultures, 4% paraformaldehyde (PFA) for 10-15 minutes at room temperature generally preserves both GPER1 antigenicity and fluorescence. Avoid methanol fixation as it can disrupt membrane protein conformation.

• Permeabilization considerations: Since GPER1 has both membrane and cytoplasmic domains, a mild permeabilization with 0.1-0.2% Triton X-100 (5-10 minutes) or 0.1% saponin is recommended to allow antibody access to all receptor populations.

• Blocking strategy: A blocking step using 5-10% normal serum (from the species not related to the primary antibody) with 1% BSA in PBS for 30-60 minutes reduces background and enhances signal specificity.

• Preserving phosphorylation status: When studying GPER1 activation states, include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) in all buffers to maintain phosphorylation of downstream signaling molecules like MST1 and YAP .

• Counterstaining options: DAPI nuclear counterstain pairs effectively with FITC for co-localization studies, as demonstrated in experiments showing GPER1 agonist G1-induced nuclear translocation of YAP .

What controls are essential when designing experiments with FITC-conjugated GPER1 antibodies?

A robust experimental design requires comprehensive controls:

• Isotype control: Include a FITC-conjugated antibody of the same isotype, concentration, and host species (rabbit polyclonal in this case) but irrelevant specificity to identify non-specific binding .

• Pharmacological controls: Include samples treated with GPER1-specific agonist G1 and antagonist G15 to verify functional responses. For example, G1 treatment induces distinctive cellular responses that should correlate with GPER1 expression patterns, while G15 should block these effects .

• Autofluorescence control: Unstained samples help determine natural cellular autofluorescence in the FITC channel, particularly important in tissues with high endogenous fluorescence like liver or brain.

• Absorption controls: Pre-absorption of the antibody with the immunizing peptide should eliminate specific staining.

• Genetic controls: Where possible, include samples with GPER1 knockdown or from GPER1 knockout models as definitive negative controls.

• Signal specificity controls: For colocalization studies, single-color controls help establish bleed-through parameters and set appropriate imaging thresholds.

How can FITC-conjugated GPER1 antibodies be optimally used to study receptor internalization and trafficking?

Studying GPER1 internalization and trafficking requires specialized approaches:

• Live-cell imaging protocol: For real-time trafficking studies, maintain cells in phenol red-free medium (phenol red has weak estrogenic activity) supplemented with 10-25 mM HEPES buffer (pH 7.4) for imaging outside CO2 incubators.

• Synchronized internalization: Treat cells with GPER1 agonist G1 (1 μM) to trigger receptor internalization . Time-lapse imaging at 1-2 minute intervals for up to 60 minutes captures the dynamic process.

• Co-visualization strategies: Combine FITC-GPER1 antibody with markers for specific endocytic compartments (early endosomes: EEA1; recycling endosomes: Rab11; lysosomes: LAMP1) using compatible fluorophores (e.g., red or far-red) to track the internalization pathway.

• Quantification approach: Employ quantitative image analysis measuring plasma membrane/cytoplasmic fluorescence intensity ratios over time, or track individual vesicles using particle tracking algorithms.

• Temperature manipulation: Compare internalization kinetics at 37°C versus 4°C (which blocks active internalization) to distinguish between active and passive processes.

• Inhibitor studies: Use specific inhibitors of different endocytic pathways (e.g., dynasore for dynamin-dependent endocytosis; filipin for caveolae-mediated endocytosis) to characterize the mechanism of GPER1 internalization.

What methodological approaches are recommended for studying GPER1 interactions with integrin signaling?

Investigation of GPER1-integrin signaling connections requires specialized techniques:

• Co-immunoprecipitation protocol: Lyse cells in a non-denaturing buffer (typically containing 1% NP-40 or Triton X-100, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, and protease/phosphatase inhibitors). Immunoprecipitate GPER1 and probe for co-precipitating integrin α5β1 or vice versa.

• Proximity ligation assay (PLA): This technique detects protein interactions that occur within 40 nm. Use GPER1 antibody and integrin α5β1 antibodies from different species, followed by species-specific secondary antibodies coupled to complementary oligonucleotides that generate a fluorescent signal when in close proximity.

• FRET analysis setup: For live interaction studies, combine FITC-labeled GPER1 antibody with a compatible FRET acceptor fluorophore-labeled integrin antibody (e.g., Cy3 or TRITC) and measure energy transfer efficiency.

• Functional validation: Treat cells with G1 and measure integrin activation using conformation-specific antibodies that recognize active integrin conformations, or assess downstream functional outcomes such as fibronectin matrix assembly .

• Genetic manipulation approach: Perform knockdown/knockout studies targeting key components of both pathways (GPER1, integrin α5β1, Src kinases) to establish interaction dependencies.

• Quantitative adhesion assays: Compare cell adhesion to fibronectin under conditions of GPER1 activation (G1 treatment) versus inhibition (G15 treatment) to functionally assess integrin activation .

How can researchers accurately quantify GPER1-mediated effects on cell survival and apoptosis?

Quantification of GPER1 effects on cell survival requires multi-parametric approaches:

• Flow cytometry protocol: Use Annexin V/propidium iodide (PI) dual staining to distinguish between early apoptotic (Annexin V+/PI-) and late apoptotic/necrotic (Annexin V+/PI+) populations following treatment with GPER1 agonists like G1 .

• Western blot analysis: Examine cleaved PARP (indicator of apoptosis) and phosphorylation status of pro-survival signaling molecules. Important targets include phosphorylated MST1 and YAP, which decrease following G1 treatment and correlate with increased cell survival .

• qRT-PCR assessment: Measure expression of:

  • Pro-survival genes: CTGF, CYR61, and ANKRD1 (increase with G1 treatment)

  • Pro-apoptotic genes: PUMA and Bim (decrease with G1 treatment)

• Mitochondrial integrity analysis: Assess mitochondrial membrane potential using JC-1 or TMRM dyes, as GPER1 activation protects against mitochondrial permeability transition pore (mPTP) opening .

• Cell cycle analysis: Quantify cell cycle distribution using PI staining of DNA content, as G1 treatment induces G2/M arrest in certain cell types such as MCF-7 .

• Caspase-independent mechanisms: Since GPER1 can induce caspase-independent cell death, evaluate PARP cleavage patterns and employ pan-caspase inhibitors (zVAD-fmk) to distinguish between caspase-dependent and independent pathways .

What are the recommended approaches for studying GPER1 effects on calcium signaling?

Investigating GPER1-mediated calcium signaling requires specialized techniques:

• Real-time calcium imaging protocol: Load cells with ratiometric calcium indicators like Indo-1 AM (as used in source material) or Fura-2 AM . Calculate baseline Ca2+ levels before adding G1 (1-5 μM), then monitor fluorescence ratio changes over time (typically 5-15 minutes).

• Calcium source determination: Use specific inhibitors to identify calcium sources:

  • Thapsigargin (TG) to deplete ER calcium stores

  • Calcium-free extracellular buffer with EGTA to eliminate external calcium contribution

  • Specific channel blockers (L-type, T-type Ca2+ channel blockers) to identify channel involvement

• Subcellular calcium dynamics: Employ organelle-targeted calcium indicators (e.g., ER-GCaMP) to track compartment-specific calcium changes in response to GPER1 activation.

• Downstream signaling assessment: Measure activation of calcium-dependent enzymes such as CaMKII through phospho-specific antibodies or FRET-based activity reporters.

• High-throughput approach: For screening studies, use plate reader-based calcium flux assays with fluorescent indicators in 96- or 384-well formats to test multiple conditions simultaneously.

• Correlation analysis: Link calcium signaling parameters (amplitude, duration, oscillation frequency) with downstream functional outcomes such as changes in gene expression or cell survival.

What are common issues with FITC-conjugated antibody detection and their solutions?

Researchers often encounter several challenges with FITC-conjugated antibodies:

• Photobleaching mitigation: FITC is relatively prone to photobleaching. Countermeasures include:

  • Adding anti-fade agents (e.g., ProLong Gold, SlowFade) to mounting media

  • Reducing exposure time and light intensity during imaging

  • Capturing FITC channel images first in multi-channel experiments

  • Using oxygen scavengers in live-cell imaging buffers

• Autofluorescence reduction: Cellular autofluorescence often overlaps with FITC emission. Solutions include:

  • Brief treatment with 0.1% Sudan Black B in 70% ethanol after immunostaining

  • Using spectral unmixing during image acquisition on confocal systems

  • Employing narrow bandpass filters to isolate specific FITC emission wavelengths

  • Considering time-gated detection as FITC fluorescence has longer lifetime than autofluorescence

• pH sensitivity management: FITC fluorescence is pH-dependent (optimal at pH 8.0). Ensure all buffers are properly pH-adjusted, especially for quantitative comparisons.

• Signal amplification options: For weak signals, consider:

  • Tyramide signal amplification (TSA) systems compatible with FITC

  • Extended antibody incubation times (overnight at 4°C)

  • Optimizing the antibody concentration through titration experiments

• Fixation artifacts: Overfixation can mask epitopes. Test shortened fixation times or epitope retrieval methods (heat-induced or enzymatic) if signal is weak.

How can researchers resolve conflicting results when studying GPER1 signaling pathways?

Addressing contradictory findings in GPER1 research requires systematic investigation:

• Cell type variation analysis: Different cell types show variable GPER1 expression and signaling outcomes. Compare results across multiple cell lines (e.g., MCF-7, SK-BR-3, MDA-MB-231) and primary cells to establish general principles versus cell-specific effects.

• Concentration-dependent effects: Test multiple G1 concentrations (1-5 μM range as in the literature) as concentration-dependent effects are common . Note that while 3-6 nM is the reported IC50 for G1, many studies use higher concentrations.

• Temporal dynamics assessment: Examine both acute (minutes to hours) and chronic (24-72 hours) responses to GPER1 activation, as signaling pathways evolve over time .

• Pathway interaction mapping: Use specific inhibitors of intersecting pathways to delineate signaling crosstalk:

  • SP600125 for JNK inhibition

  • SB203580 for p38 MAPK inhibition

  • GSK2606414 for PERK inhibition
    These help resolve whether observed effects are direct or indirect .

• Genetic verification: Confirm pharmacological results with genetic approaches (siRNA, CRISPR) targeting GPER1 to rule out off-target effects of G1 at high concentrations.

• Experimental condition standardization: Control for variables that affect estrogen signaling:

  • Use phenol red-free media (phenol red has weak estrogenic activity)

  • Charcoal-stripped serum to remove endogenous steroids

  • Consistent timing relative to cell cycle phase

How can FITC-conjugated GPER1 antibodies be utilized in multiplexed imaging systems?

Multiplexed imaging with FITC-GPER1 antibodies enables comprehensive pathway analysis:

• Spectral compatibility planning: FITC (excitation ~495 nm, emission ~519 nm) pairs effectively with:

  • Red fluorophores (e.g., Cy3, TRITC) for double labeling

  • Far-red fluorophores (e.g., Cy5, Alexa 647) and blue nuclear stains (DAPI) for triple labeling

• Sequential staining protocol: For complex multiplexing with antibodies from the same species:

  • First round: FITC-GPER1 antibody, image

  • Quenching step: Use 0.1-1% H2O2 or specific antibody elution buffers

  • Subsequent rounds: Additional primary-secondary pairs

  • Digital overlay of sequentially acquired images

• Multispectral imaging approach: Use systems with spectral detectors to separate closely overlapping fluorophores through spectral unmixing algorithms.

• Co-localization analysis: Employ quantitative co-localization with key pathway components:

  • YAP nuclear translocation (as shown in response to G1 treatment)

  • Integrin α5β1 clustering at adhesion sites

  • EGFR co-localization during transactivation events

• Spatial profiling integration: Combine with spatial transcriptomics or mass cytometry imaging to correlate protein localization with transcriptional outputs or other protein markers.

What methodological approaches should be used to investigate GPER1's role in mitochondrial function?

Investigation of GPER1-mitochondrial connections requires specialized techniques:

• Mitochondrial isolation protocol: For biochemical studies, isolate intact mitochondria using differential centrifugation with sucrose buffer. Verify fraction purity using markers (VDAC, cytochrome c for mitochondria; calnexin for ER; β-actin for cytosol) .

• Mitochondrial membrane potential assessment: Use fluorescent indicators (TMRM, JC-1) to measure membrane potential changes following GPER1 activation or inhibition. Quantify by flow cytometry or live-cell imaging.

• Mitochondrial permeability transition pore (mPTP) opening assay: Load cells with calcein-AM and CoCl2 quencher. mPTP opening allows Co2+ entry into mitochondria, quenching calcein fluorescence. G1 treatment can be assessed for protective effects against mPTP opening .

• Bioenergetic profiling: Measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using Seahorse XF analyzers to assess effects of GPER1 modulation on mitochondrial respiration and glycolysis.

• Mitochondrial morphology analysis: Perform confocal microscopy with mitochondrial stains (MitoTracker) following G1 treatment. Quantify morphological parameters (length, branching, circularity) using software like Imaris or MiNA.

• mtDNA/nuclear DNA ratio determination: Extract DNA and perform qPCR for mitochondrial genes (e.g., MT-ND1) and nuclear genes (e.g., β-actin) to determine if GPER1 activation affects mitochondrial biogenesis over time .