PTGER3 Antibody, HRP conjugated

What is PTGER3 and what biological functions does it regulate?

PTGER3 is the receptor for prostaglandin E2 (PGE2) and plays diverse physiological roles throughout the body. This receptor's activity involves dual signaling mechanisms: inhibition of adenylate cyclase mediated by G-I proteins and elevation of intracellular calcium levels . PTGER3 plays crucial roles in multiple biological processes including inhibition of gastric acid secretion, modulation of neurotransmitter release in central and peripheral neurons, inhibition of sodium and water reabsorption in kidney tubules, and contraction of uterine smooth muscle . Research has also demonstrated PTGER3's involvement in fever development in response to pyrinogens (including IL1B, prostaglandin E2, and bacterial lipopolysaccharide), potentiation of platelet aggregation, and protection of duodenal mucosa against acid-induced ulceration .

What are the technical specifications of commercially available PTGER3 Antibody, HRP conjugated?

Commercial HRP-conjugated PTGER3 antibodies typically have the following specifications:

• Reactivity: Human-specific in most products

• Host: Typically raised in rabbit

• Isotype: IgG

• Clonality: Both polyclonal and monoclonal options available, with polyclonal being more common

• Storage conditions: -20°C or -80°C, avoiding repeated freeze-thaw cycles

• Storage buffer: Typically contains preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4)

• Purification method: Protein G purification with >95% purity

• Immunogen: Commonly derived from recombinant Human Prostaglandin E2 receptor EP3 subtype protein (amino acids 1-49)

What techniques are compatible with PTGER3 Antibody, HRP conjugated?

• Western blot: Successfully detecting PTGER3 in human Hodgkin's lymphoma cell line (HDLM-2) and kidney tissue with a specific band at approximately 53 kDa

• Immunohistochemistry (paraffin-embedded sections): Detecting PTGER3 in human kidney tissue with specific cytoplasmic localization in convoluted tubules

• Immunocytochemistry/Immunofluorescence: Suitable for cellular localization studies

For applications beyond ELISA, researchers may need to validate the HRP-conjugated antibody or consider secondary antibody approaches.

How should I design an optimal ELISA protocol using PTGER3 antibody, HRP conjugated?

When designing an ELISA protocol with HRP-conjugated PTGER3 antibody, consider the following methodological approach:

• Plate preparation: Coat high-binding 96-well plates with recombinant PTGER3 protein or sample containing PTGER3 (overnight at 4°C).

• Blocking: Block non-specific binding sites with 2-5% BSA or non-fat milk in PBS or TBS (1-2 hours at room temperature).

• Primary antibody incubation: Since the PTGER3 antibody is already HRP-conjugated, dilute to manufacturer-recommended concentration (typically 1:1000 to 1:5000) in blocking buffer with 0.05% Tween-20 .

• Washing: Perform 3-5 wash steps with PBS-T or TBS-T (containing 0.05-0.1% Tween-20).

• Substrate addition: Add appropriate HRP substrate (TMB, ABTS, or OPD) and monitor color development.

• Signal detection: Stop the reaction if necessary and measure absorbance at the appropriate wavelength.

For optimal results, perform multiple dilutions of both samples and antibody to establish a standard curve and determine the linear range of detection. Cross-validation with a non-conjugated PTGER3 antibody may provide additional confidence in results.

What controls should be included when using PTGER3 antibody, HRP conjugated?

A rigorous experimental design should include the following controls:

• Positive control: Lysates from HDLM-2 human Hodgkin's lymphoma cell line or human kidney tissue extracts, which have been verified to express PTGER3 .

• Negative control: Samples known to lack PTGER3 expression.

• Isotype control: A non-specific rabbit IgG-HRP conjugate at the same concentration to assess non-specific binding .

• Blocking peptide control: Pre-incubation of the antibody with the immunogen peptide (1-49AA of human PTGER3) should abolish specific signal.

• Secondary-only control: For comparison experiments with non-conjugated antibodies.

• Substrate-only control: To establish background signal from the detection system.

These controls help distinguish specific signal from non-specific background and validate experimental findings.

How can I optimize antigen retrieval for immunohistochemistry studies of PTGER3?

While HRP-conjugated PTGER3 antibodies are primarily designed for ELISA, researchers utilizing non-conjugated PTGER3 antibodies for IHC should consider these antigen retrieval parameters:

• Heat-induced epitope retrieval (HIER): Use basic antigen retrieval buffer (pH 9.0) as reported in successful detection protocols . Heat samples to 95-100°C for 20 minutes in a water bath, pressure cooker, or microwave.

• Enzyme-based retrieval: Alternative approach using proteinase K (10-20 μg/ml) for 10-15 minutes at room temperature.

• Incubation parameters: Following retrieval, 0.1 μg/mL of antibody for 1 hour at room temperature has proven effective for detecting PTGER3 in kidney tissue .

• Detection system: For non-conjugated antibodies, polymer-based detection systems (e.g., Anti-Mouse IgG VisUCyte HRP Polymer) have demonstrated superior results compared to traditional ABC methods .

The specific localization pattern in kidney tissue (cytoplasmic staining in convoluted tubules) serves as a valuable positive control for optimizing these parameters .

How can I resolve weak or absent signal when using PTGER3 antibody, HRP conjugated?

When troubleshooting weak or absent signal, consider these methodological adjustments:

• Antibody concentration: Increase antibody concentration incrementally, testing a range from 1:500 to 1:2000 .

• Antigen abundance: PTGER3 expression varies across tissues; kidney tissue and HDLM-2 cells demonstrate detectable levels .

• Blocking optimization: Test alternative blocking agents (BSA, casein, commercial blockers) as excessive blocking can mask epitopes.

• Incubation conditions: Extended incubation at 4°C (overnight) may improve binding kinetics compared to shorter room temperature incubations.

• Detection sensitivity: For weak signals, consider switching to more sensitive HRP substrates like SuperSignal West Femto rather than standard ECL reagents.

• Protein denaturation: Confirm sample preparation maintains the native epitope structure, as the immunogen spans amino acids 1-49 .

• Storage conditions: HRP conjugates are sensitive to repeated freeze-thaw cycles; aliquot antibodies and store at -20°C or -80°C .

If signal remains problematic, consider validating PTGER3 expression using orthogonal methods such as qPCR before extensive antibody troubleshooting.

What could explain unexpected bands or staining patterns when using PTGER3 antibodies?

Unexpected results may stem from several technical and biological factors:

• Protein modifications: PTGER3 undergoes post-translational modifications, and the mature protein approximately 53 kDa in Western blot analysis . Bands at different molecular weights may represent:

  • Glycosylated isoforms

  • Proteolytic fragments

  • Splice variants

  • Aggregation or oligomerization

• Cross-reactivity: While PTGER3 antibodies are human-specific, potential cross-reactivity with other prostaglandin receptor family members (PTGER1, PTGER2, PTGER4) may occur due to sequence homology.

• Isoform specificity: The human PTGER3 gene produces multiple isoforms with identical ligand binding properties but different C-terminal tails and signaling capabilities . Confirm whether your antibody targets conserved (N-terminal) or variable regions.

• Non-specific binding: Optimize blocking and washing protocols to reduce background. Inclusion of 0.1-0.3% Triton X-100 in blocking buffers can reduce non-specific membrane interactions.

• Sample preparation: Protein denaturation conditions can affect epitope accessibility. Test both reducing and non-reducing conditions as demonstrated in successful Western blot protocols .

How should I interpret PTGER3 expression data in the context of prostaglandin signaling pathways?

When interpreting PTGER3 expression data, consider these analytical frameworks:

• Cellular localization: PTGER3 primarily exhibits cytoplasmic localization in convoluted tubules of kidney tissue . Altered localization patterns may suggest dysregulation or pathology.

• Co-expression analysis: PTGER3 functions within the broader prostaglandin signaling network. Analyzing co-expression with cyclooxygenases (COX-1, COX-2), other EP receptors (EP1, EP2, EP4), and downstream effectors provides more comprehensive pathway insights.

• Signaling crosstalk: PTGER3 signals through both inhibition of adenylate cyclase and elevation of intracellular calcium . Measure downstream markers (cAMP levels, calcium flux) to confirm functional activity.

• Physiological context: Interpret PTGER3 expression in relation to its diverse physiological roles:

  • In kidney: Involved in sodium and water reabsorption

  • In neural tissues: Modulates neurotransmitter release

  • In gastric tissue: Regulates acid secretion

  • In uterine tissue: Mediates smooth muscle contraction

• Pathological alterations: Changes in PTGER3 expression have been implicated in inflammation, fever response, blood coagulation, and acid-induced ulceration .

How can multiplex analysis be performed with PTGER3 antibody, HRP conjugated?

Advanced multiplex strategies with PTGER3 antibody include:

• Sequential multiplexing: For tissue sections, perform sequential stripping and re-probing:

  • Document PTGER3-HRP staining

  • Strip antibodies using glycine buffer (pH 2.2) or commercial stripping solutions

  • Verify complete stripping

  • Re-probe with antibodies against related pathway components

• Spectral unmixing approaches: Utilize multiple chromogenic substrates for HRP that produce different colored precipitates:

  • DAB (brown)

  • AEC (red)

  • TMB (blue)

  • Vector VIP (purple)

• Compartmental analysis: Combine HRP-conjugated PTGER3 antibody with fluorescent antibodies targeting distinct cellular compartments:

  • Membrane markers (Na+/K+ ATPase)

  • Nuclear markers (DAPI)

  • Organelle markers (Golgi, ER, mitochondria)

• Single-cell resolution techniques: Adapt PTGER3 detection for techniques like:

  • Mass cytometry (CyTOF)

  • Digital spatial profiling

  • Imaging mass cytometry

These approaches enable comprehensive pathway analysis beyond single-target detection.

What are the considerations for using PTGER3 antibody in studies of isoform-specific signaling?

The PTGER3 gene generates multiple splice variants with unique C-terminal domains and signaling properties . When investigating isoform-specific signaling:

• Epitope mapping: Most commercially available PTGER3 antibodies target the N-terminal domain (amino acids 1-49) , which is conserved across isoforms. For isoform-specific detection, confirm epitope specificity or generate custom antibodies against unique C-terminal sequences.

• Functional validation: Different PTGER3 isoforms couple to distinct signaling pathways. Validate antibody specificity through functional assays:

  • G-protein coupling (Gαi vs. Gαs)

  • Adenylate cyclase inhibition

  • Calcium mobilization

  • ERK/MAPK activation

• Expression systems: For controlled isoform studies, use recombinant expression systems with defined PTGER3 isoforms combined with functional readouts.

• Complementary approaches: Combine antibody-based detection with:

  • Isoform-specific PCR primers

  • RNA-seq for splice variant quantification

  • Mass spectrometry for isoform-specific peptide detection

• Knockout/knockdown validation: Utilize CRISPR/Cas9 or siRNA approaches targeting specific isoforms to validate antibody specificity.

How can PTGER3 antibody be used to investigate receptor internalization and trafficking?

Investigating PTGER3 receptor dynamics requires specialized approaches:

• Pulse-chase methodology:

  • Surface label PTGER3 receptors at 4°C

  • Allow internalization at 37°C for varying time points

  • Strip remaining surface receptors

  • Quantify internalized fraction using PTGER3 antibody

• Co-localization studies: Track PTGER3 trafficking through endocytic compartments using co-staining with:

  • Early endosome markers (EEA1)

  • Recycling endosome markers (Rab11)

  • Late endosome/lysosome markers (LAMP1)

• Live-cell imaging: For dynamic studies, consider:

  • Generating GFP-tagged PTGER3 constructs

  • Performing antibody feeding assays with fluorescently-labeled PTGER3 antibody fragments

• Biochemical fractionation: Separate membrane and cytosolic fractions to quantify receptor internalization following agonist stimulation.

• Stimulation protocols: Design time-course experiments with PGE2 stimulation to capture:

  • Rapid internalization (minutes)

  • Recycling phases (30-60 minutes)

  • Downregulation (hours)

These approaches enable precise characterization of PTGER3 receptor regulation following ligand engagement.

How can PTGER3 antibodies be integrated into single-cell analytical platforms?

Emerging single-cell technologies offer new applications for PTGER3 antibodies:

• Single-cell Western blotting: Miniaturized Western blot techniques allow protein analysis at single-cell resolution:

  • Capture cells in microwell arrays

  • Perform in-situ lysis

  • Separate proteins by size

  • Detect PTGER3 with HRP-conjugated antibody

• Microfluidic immunoassays: Droplet-based or chamber-based microfluidic systems enable:

  • Multiplex protein detection in single cells

  • Correlation of PTGER3 levels with functional outputs

  • Time-resolved signaling studies

• Mass cytometry adaptation: Metal-tagged antibodies for CyTOF analysis allow:

  • Simultaneous measurement of >40 parameters

  • Integration of PTGER3 detection into comprehensive signaling panels

  • Clustering analysis to identify distinct cell populations

• Spatial transcriptomics integration: Combine PTGER3 protein detection with spatial transcriptomics to correlate:

  • Protein expression

  • mRNA levels

  • Spatial organization within tissues

These emerging platforms enable unprecedented resolution of PTGER3 biology within heterogeneous cell populations.

What are the technical considerations for using PTGER3 antibodies in high-throughput drug screening assays?

Implementation of PTGER3 antibodies in drug discovery requires specific technical adaptations:

• Assay miniaturization: Transition from standard to high-density plate formats:

  • Optimize antibody concentration for 384/1536-well formats

  • Establish minimal detection volume requirements

  • Validate signal-to-background ratios in miniaturized format

• Automation compatibility: Ensure protocols are compatible with liquid handling systems:

  • Minimize incubation steps

  • Optimize wash procedures

  • Develop robust timing parameters

• Readout optimization: For HRP-conjugated antibodies:

  • Select substrates with extended signal stability

  • Consider chemiluminescent over colorimetric detection for wider dynamic range

  • Establish reference standards for plate-to-plate normalization

• Positive controls: Include known PTGER3 modulators as controls:

  • PGE2 as natural ligand

  • Established synthetic agonists

  • Antagonists like L-798,106

• Data analysis pipelines: Develop algorithms for:

  • Dose-response curve fitting

  • Hit selection

  • Structure-activity relationship analysis

These adaptations enable efficient screening of compound libraries for modulators of PTGER3-mediated signaling.

How might PTGER3 antibodies contribute to understanding receptor heteromerization?

Investigating PTGER3 heteromerization with other GPCRs represents a frontier application:

• Proximity-based detection methods:

  • Proximity ligation assay (PLA) using PTGER3 antibody paired with antibodies against potential heteromerization partners

  • FRET/BRET approaches with appropriately labeled antibody fragments

  • Co-immunoprecipitation followed by mass spectrometry analysis

• Functional consequences assessment:

  • Altered ligand binding properties

  • Modified signaling cascade activation

  • Changed desensitization/internalization kinetics

• Tissue-specific heteromerization patterns:

  • Brain regions (neurotransmitter modulation)

  • Kidney (water/electrolyte balance)

  • Inflammatory tissues (pain/fever regulation)

• Pathological alterations:

  • Changes in heteromerization patterns in disease states

  • Correlation with altered drug responses

  • Potential therapeutic targeting of specific heteromers