PRLR Antibody, FITC conjugated

What is PRLR and why are PRLR antibodies important in research?

PRLR (Prolactin Receptor) belongs to the cytokine type I subfamily, which includes receptors for leptin, leukemia inhibiting factor, and erythropoietin. It functions as a receptor for the anterior pituitary hormone prolactin (PRL) and exhibits significant biological importance in various physiological processes. PRLR exists in multiple isoforms, including long and short forms, with molecular weights of approximately 70 kDa and 45 kDa respectively, while the mature glycosylated form migrates at 85-95 kDa .

PRLR antibodies are crucial research tools because they enable:

• Detection and quantification of receptor expression in normal and pathological tissues

• Investigation of receptor trafficking and internalization pathways

• Study of prolactin-mediated signaling events

• Evaluation of receptor status in cancer tissues, particularly breast cancer

• Analysis of structure-function relationships of different receptor isoforms

PRLR acts as a prosurvival factor for spermatozoa by inhibiting sperm capacitation through suppression of SRC kinase activation and stimulation of AKT. Certain isoforms (specifically isoform 4 and isoform 6) are unable to transduce prolactin signaling .

What are the major PRLR isoforms detected by commercially available antibodies?

Commercial PRLR antibodies detect several isoforms with distinct biological properties:

Antibody clone 1A2B1 reacts with the long form of human PRLR (hPRLr) at ~85-90 kDa and may identify intermediate and delta S1 isoforms by western blotting. On immunoprecipitation, this antibody strongly recognizes only the long form of hPRLr, with reactivity confirmed in human T-47D breast carcinoma cells during development .

Clone 67292-1-Ig detects multiple forms (45 kDa, 70 kDa, and 85-95 kDa) and has been tested for reactivity with human and mouse samples across multiple cell lines including Jurkat, A431, T-47D, and 4T1 cells .

How does FITC conjugation of PRLR antibodies enhance research applications?

FITC conjugation of PRLR antibodies provides several methodological advantages:

• Direct detection: Eliminates the need for secondary antibodies, reducing background and cross-reactivity issues.

• Multiparametric analysis: Enables simultaneous detection of multiple targets when combined with other fluorophore-conjugated antibodies.

• Internalization studies: Facilitates real-time visualization of receptor trafficking.

• Flow cytometry applications: Provides high sensitivity detection of surface and internalized receptors.

For internalization studies, researchers commonly use FITC-conjugated secondary antibodies with PRLR primary antibodies. For example, in receptor internalization assays, cells expressing HA-tagged PRLr are allowed to internalize anti-HA antibody complexed with FITC-conjugated goat anti-mouse Fab for tracking the internalization process .

What cell lines are recommended for validating PRLR antibody specificity?

Based on the provided research data, the following cell lines have demonstrated reliable PRLR expression and are suitable for antibody validation:

T-47D breast carcinoma cells are particularly valuable as they express high levels of PRLR and have been consistently used to validate antibody reactivity during development . When conducting validation experiments, it is advisable to include both positive and negative control cell lines to confirm antibody specificity.

How should researchers optimize PRLR antibody dilutions for different applications?

Optimal dilution of PRLR antibodies varies by application technique and specific antibody clone. A methodological approach should include:

Western Blotting Optimization:

• Start with manufacturer-recommended dilutions (typically 1:500-1:2000 for PRLR antibodies)

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Evaluate signal-to-noise ratio at each concentration

• Select the dilution that provides clear bands with minimal background

• Consider blocking optimization (5% non-fat milk or BSA) to reduce non-specific binding

Immunohistochemistry Protocol:

• Begin with recommended dilutions (1:200-1:500 for many PRLR antibodies)

• Test multiple antigen retrieval methods (both citrate buffer pH 6.0 and TE buffer pH 9.0 have been successful with PRLR antibodies)

• Systematically evaluate multiple parameters:

  • Primary antibody incubation time (1 hour vs. overnight)

  • Incubation temperature (room temperature vs. 4°C)

  • Detection system sensitivity

Flow Cytometry Applications:

• Initial testing at 60 nmol/L concentration for cell binding studies

• Titration to determine optimal saturation concentration

• Include isotype controls at matching concentrations

Each testing system requires optimization, as antibody performance is often sample-dependent. Document optimal conditions for reproducibility across experiments .

What protocol is recommended for studying PRLR internalization using FITC-conjugated antibodies?

For studying PRLR internalization, researchers have successfully employed the following flow cytometry-based protocol:

Materials Required:

• Target cells expressing PRLR (e.g., T47D cells)

• PRLR antibody or anti-tag antibody (if using tagged PRLR)

• FITC-conjugated secondary antibody

• Fixation solution (2% paraformaldehyde)

• Appropriate buffers (PBS, cell culture medium)

Detailed Protocol:

• Incubate cells with PRLR antibody (or control ADCs at 60 nmol/L) on ice for 1 hour

• Wash cells to remove unbound antibodies

• Set aside an aliquot of cells to remain on ice (0-time point control)

• Incubate remaining cells at 37°C for different time periods (e.g., 1, 2, 4, and 6 hours)

• Fix all samples in 2% paraformaldehyde for 20 minutes at room temperature

• Stain with FITC-labeled secondary antibody (if using unconjugated primary)

• Analyze by flow cytometry

• Calculate internalization percentage using the formula:
  % internalization = (MFIice - MFI37°C)/MFIice × 100%

This method allows for quantitative assessment of receptor internalization kinetics. For more detailed trafficking analysis, this approach can be complemented with confocal microscopy .

How can researchers overcome cross-reactivity issues with PRLR antibodies?

Cross-reactivity remains a significant challenge when working with PRLR antibodies. Researchers can implement these methodological approaches:

• Validate using multiple antibody clones: Compare results from different antibodies targeting distinct PRLR epitopes

• Implement genetic controls:

  • Use PRLR knockout cell lines as negative controls

  • Compare with PRLR-overexpressing systems as positive controls

• Pre-absorption testing: Pre-incubate antibody with purified PRLR protein to confirm specificity

• Cross-species validation: Test reactivity in predicted reactive species (many PRLR antibodies show reactivity with human, mouse, and rat)

• Immunoprecipitation confirmation: Follow western blotting with immunoprecipitation to verify specificity to particular isoforms

For immunohistochemistry applications, careful optimization of antigen retrieval methods is critical. Both TE buffer (pH 9.0) and citrate buffer (pH 6.0) have been successful with different PRLR antibodies and tissue types .

What are the recommended protocols for using PRLR antibodies in affinity measurement studies?

Surface Plasmon Resonance (SPR) has been successfully employed to determine the binding affinity of PRLR antibodies. A methodological approach includes:

Materials Required:

• Purified recombinant PRLR protein

• PRLR antibody (purified)

• SPR instrument with CM5 sensor chip

• Appropriate buffers (running buffer, regeneration buffer)

Protocol for SPR Affinity Measurement:

• Couple human PRLR-Fc to CM5 sensor chip

• Inject PRLR antibody at various concentrations

• Allow association for approximately 100 seconds

• Monitor dissociation over an appropriate time period

• For bispecific antibodies, demonstrate simultaneous binding by sequential injection of second antigen (e.g., HER2-Fc at 50 nmol/L)

• Use appropriate controls (PBS injection)

• Calculate binding kinetics (kon, koff) and equilibrium dissociation constant (KD)

This approach has been successfully used to characterize bispecific antibodies targeting PRLR and HER2, demonstrating retained affinity after drug conjugation .

How are PRLR antibodies being utilized in cancer-targeted therapeutic approaches?

PRLR antibodies have shown considerable potential in cancer research, particularly for targeted therapies:

Antibody-Drug Conjugates (ADCs):
Researchers have developed ADCs utilizing PRLR antibodies to selectively deliver cytotoxic agents to cancer cells. For example, conjugating monomethyl auristatin E (MMAE) to PRLR antibodies via a cleavable linker enables targeted delivery of this potent microtubule-disrupting agent .

Bispecific Antibody Drug Conjugates (BsADCs):
Advanced approaches include bispecific antibodies targeting both PRLR and HER2, which have demonstrated:

• Enhanced internalization efficiency compared to monospecific HER2-targeting ADCs

• Superior antitumor activity in vitro

• Potential to overcome resistance mechanisms through dual-receptor targeting

These bispecific constructs are generated through platforms such as "Bispecific Antibody by Protein Trans-splicing (BAPTS)" and conjugated with payloads like MMAE .

Internalization-Dependent Efficacy:
The efficacy of these therapeutic approaches depends heavily on receptor internalization, with studies demonstrating that:

• PRLR undergoes polyubiquitination-stimulated internalization

• Receptor trafficking can be manipulated to enhance therapeutic delivery

• Internalization kinetics directly impact therapeutic efficacy

This research area represents a promising frontier for treating cancers with elevated PRLR expression, including breast cancer.

What method is recommended for distinguishing between different PRLR isoforms in complex samples?

Distinguishing between PRLR isoforms requires a multifaceted approach:

Comprehensive Isoform Detection Protocol:

• Sample preparation optimization:

  • For membrane proteins: Use specialized lysis buffers containing 1% n-octyl β-d-glucopyranoside and 0.1% SDS

  • Include protease inhibitor cocktail to prevent degradation

• SDS-PAGE separation:

  • Use gradient gels (7.5-10%) for optimal separation of various isoform molecular weights

  • Long form: 85-90 kDa

  • Intermediate forms: Variable

  • Short form: ~45 kDa

• Western blot detection:

  • Select antibodies validated for specific isoform detection

  • Clone 1A2B1 recognizes long form (85-90 kDa) and potentially intermediate forms

  • Clone 67292-1-Ig detects multiple forms (45, 70, 85-95 kDa)

• Confirmationary assays:

  • Immunoprecipitation to isolate specific isoforms

  • RT-PCR with isoform-specific primers to correlate protein detection with mRNA expression

• Control samples:

  • Include tissues/cells with known isoform expression patterns

  • Consider recombinant expression of specific isoforms as positive controls

This methodological approach enables researchers to reliably distinguish between functionally distinct PRLR variants in complex biological samples.

How can researchers effectively quantify PRLR surface expression and internalization kinetics?

Quantitative analysis of PRLR surface expression and internalization requires rigorous methodology:

Surface Expression Quantification:

• Flow cytometry protocol:

  • Label cells with unconjugated PRLR antibody followed by FITC-conjugated secondary antibody

  • Alternatively, use directly conjugated PRLR antibodies

  • Include calibration beads with known antibody binding capacity

  • Convert mean fluorescence intensity to molecules per cell

• Cell surface biotinylation assay:

  • Biotinylate cell surface proteins using Sulfo-NHS-SS-Biotin

  • Lyse cells in buffer containing 0.5% Triton X-100, 0.1% n-octyl β-d-glucopyranoside, and 0.1% SDS

  • Recover biotinylated proteins using immobilized NeutrAvidin resins

  • Detect PRLR by western blotting

  • Quantify using densitometry and appropriate software (e.g., Image software)

Internalization Kinetics Measurement:

• Antibody-based tracking:

  • Incubate cells with anti-PRLR or anti-tag antibody on ice (prevents internalization)

  • Allow internalization by warming to 37°C for various time points

  • Quantify surface-remaining vs. internalized receptor using FITC-conjugated secondary antibodies

  • Calculate percent internalization using formula: % internalization = (MFIice - MFI37°C)/MFIice × 100%

• Recycling assay:

  • Track reappearance of internalized receptor-antibody complexes on cell surface

  • Quantify using methods like Amplex Red assay after secondary HRP-conjugated labeling

  • Plot percentage of internalized receptor that recycles to surface over time

These approaches provide complementary quantitative data on PRLR dynamics, crucial for understanding receptor biology and designing effective therapeutic strategies.

What considerations are important when designing experiments to study PRLR-mediated signaling pathways?

Investigating PRLR-mediated signaling requires careful experimental design:

Critical Experimental Design Considerations:

• Isoform-specific effects:

  • Different PRLR isoforms exhibit distinct signaling capabilities

  • Long form (85-90 kDa) is signaling-competent

  • Isoform 4 and isoform 6 are unable to transduce prolactin signaling

  • Selective detection or expression is essential for pathway delineation

• Pathway-specific readouts:

  • JAK/STAT pathway: Monitor STAT5 phosphorylation

  • MAPK pathway: Assess ERK1/2 phosphorylation

  • PI3K/AKT pathway: Evaluate AKT phosphorylation

  • Include appropriate time-course analysis (rapid vs. sustained signaling)

• Receptor modification monitoring:

  • Ubiquitination: Detect with anti-ubiquitin antibodies (e.g., FK2)

  • Phosphorylation: Use phospho-specific antibodies

  • Consider immunoprecipitation to isolate receptor complexes

• Antagonist controls:

  • Include PRL antagonists as negative controls

  • Design concentration-response experiments

  • Account for receptor desensitization in prolonged stimulation protocols

• Cross-talk analysis:

  • Consider PRLR interaction with other receptors (e.g., HER2)

  • Study signaling in the context of receptor co-expression

  • Evaluate synergistic or antagonistic pathway interactions

These methodological considerations ensure robust and reproducible analysis of complex PRLR signaling networks in various physiological and pathological contexts.

How should researchers address contradictory results when using different PRLR antibody clones?

When confronted with discrepancies between different PRLR antibody clones, a systematic troubleshooting approach is essential:

Resolution Protocol for Antibody Discrepancies:

• Epitope mapping analysis:

  • Determine the specific epitopes recognized by each antibody

  • Different clones may recognize distinct domains (extracellular vs. intracellular)

  • Epitope accessibility may vary depending on sample preparation

  • Clone 1A2B1 targets the extracellular domain , while other clones may target different regions

• Isoform specificity verification:

  • Confirm which PRLR isoforms each antibody detects

  • Some antibodies preferentially detect long forms (85-90 kDa)

  • Others may detect multiple forms including short variants (45 kDa)

  • Validate using recombinant expression of specific isoforms

• Application-specific optimization:

  • Different antibodies may perform optimally in different applications

  • Some may work well for western blotting but poorly for immunohistochemistry

  • Optimize conditions independently for each antibody clone

  • For western blots: Test multiple blocking agents (5% milk vs. BSA)

  • For immunohistochemistry: Compare antigen retrieval methods (citrate buffer pH 6.0 vs. TE buffer pH 9.0)

• Validation through complementary techniques:

  • Confirm protein expression using mRNA analysis

  • Implement PRLR knockdown/knockout controls

  • Use functional assays to correlate with receptor detection

• Data integration strategy:

  • Report results with multiple antibodies when discrepancies exist

  • Consider the collective evidence rather than relying on a single antibody

  • Document all variables that might influence antibody performance

What controls are essential for validating PRLR antibody specificity in immunofluorescence studies?

Rigorous validation of PRLR antibodies for immunofluorescence requires comprehensive controls:

Essential Control Panel:

• Positive control tissues/cells:

  • T-47D breast cancer cells (known high PRLR expression)

  • Human breast cancer tissue (validated for PRLR detection)

  • Tissues with physiological PRLR expression (mammary gland, ovary)

• Negative control tissues/cells:

  • Cell lines with minimal PRLR expression (validated by other methods)

  • PRLR knockout/knockdown models

  • Non-relevant tissues (based on tissue expression databases)

• Antibody-specific controls:

  • Isotype control antibodies at matching concentrations

  • Pre-absorption with recombinant PRLR protein

  • Peptide competition assays with immunizing peptides

  • Secondary antibody-only controls to assess background

• Signal validation controls:

  • Concentration gradient of primary antibody

  • Multiple fixation methods comparison (paraformaldehyde, methanol)

  • Subcellular localization consistent with known receptor biology

  • Co-localization with established cellular markers

• Cross-validation experiments:

  • Compare results from multiple antibody clones

  • Correlate with other detection methods (western blot, flow cytometry)

  • Verify expression changes under conditions known to regulate PRLR

Implementation of these controls ensures confidence in the specificity and reliability of PRLR detection in immunofluorescence applications.

How can researchers optimize protocols for detecting low-abundance PRLR in challenging samples?

Detecting low-abundance PRLR requires enhanced sensitivity protocols:

Signal Amplification Strategies:

• Western blotting optimization:

  • Increase protein loading (up to 50-100 μg total protein)

  • Use high-sensitivity chemiluminescent substrates

  • Employ signal enhancers compatible with antibody detection systems

  • Optimize antibody concentration (typically 1:500-1:2000 for PRLR antibodies)

  • Consider membrane transfer efficiency (PVDF vs. nitrocellulose)

• Immunoprecipitation enrichment:

  • Pre-enrich target protein before detection

  • Some antibodies (like 1A2B1) are validated for immunoprecipitation

  • Use optimized lysis buffers with appropriate detergents

  • Implement extended incubation times (overnight at 4°C)

• Immunohistochemistry signal enhancement:

  • Employ polymer-based detection systems

  • Use tyramide signal amplification

  • Optimize antigen retrieval (both pH 6.0 citrate and pH 9.0 TE buffers have been effective)

  • Extend primary antibody incubation (overnight at 4°C)

  • Increase antibody concentration within validated range (1:200-1:500)

• Flow cytometry sensitivity improvement:

  • Use fluorophores with higher quantum yield

  • Implement indirect staining with biotinylated secondaries and streptavidin-fluorophore

  • Analyze larger cell numbers (≥100,000 events)

  • Optimize instrument voltage settings for maximum signal separation

• Receptor upregulation strategies:

  • Pre-treat samples with agents known to upregulate PRLR

  • Consider hormonal manipulation to increase receptor expression

  • Use positive control cell lines with documented expression levels

These methodological refinements enable detection of PRLR even in challenging samples with naturally low abundance or in tissues with high background.

What are the key considerations for preserving PRLR epitopes during sample preparation for antibody-based detection?

Preserving PRLR epitopes requires careful attention to sample preparation:

Epitope Preservation Protocol:

• Tissue fixation optimization:

  • For PRLR detection, 10% neutral buffered formalin provides good epitope preservation

  • Limit fixation time (24-48 hours maximum)

  • Consider gentler fixatives for sensitive epitopes

  • For frozen sections, use freshly prepared 4% paraformaldehyde

• Membrane protein extraction:

  • Use specialized lysis buffers containing:

    • 0.5% Triton X-100

    • 0.1% n-octyl β-d-glucopyranoside

    • 0.1% SDS in HBS

  • Include protease inhibitor cocktail

  • Maintain cold temperature throughout extraction (on ice)

  • Process samples immediately after collection when possible

• Antigen retrieval optimization:

  • For PRLR antibodies, test both:

    • TE buffer pH 9.0 (suggested primary method)

    • Citrate buffer pH 6.0 (alternative method)

  • Optimize heat treatment duration (typically 20-30 minutes)

  • Allow gradual cooling to enhance epitope recovery

  • For each antibody clone, determine optimal retrieval conditions

• Storage considerations:

  • Store antibodies at -20°C with 50% glycerol pH 7.3

  • Maintain stability for one year after shipment

  • For 20μl sizes containing 0.1% BSA, avoid repeated freeze-thaw cycles

  • For tissue samples, minimize storage time before processing

• Processing controls:

  • Include positive control samples processed identically

  • Process samples consistently across experimental groups

  • Document all procedural details for reproducibility

These methodological considerations ensure optimal preservation of PRLR epitopes, maximizing detection sensitivity and specificity across various experimental applications.