Recombinant Rat Prolactin protein (Prl) (Active)

How do expression systems impact the properties of recombinant rat Prolactin protein?

Recombinant rat Prolactin is commonly expressed in Escherichia coli bacterial systems, which produces a protein with high purity (>95-97%) but lacks eukaryotic post-translational modifications . This expression system difference is significant because:

• E. coli-expressed Prolactin lacks glycosylation patterns present in native Prolactin

• The recombinant protein may have different folding characteristics

• Endotoxin levels must be controlled (<1 EU/μg or >0.05 EU/μg depending on preparation method)

When using E. coli-expressed Prolactin, researchers should consider these factors when interpreting results, especially when comparing to effects of native Prolactin in biological systems.

What are the critical quality parameters for evaluating recombinant rat Prolactin preparations?

Key quality parameters researchers should assess include:

Researchers should verify these parameters before experimental use, as variation can significantly impact experimental outcomes. SDS-PAGE analysis under reducing and non-reducing conditions can provide critical information about protein integrity and potential oligomeric states .

What are the optimal reconstitution and storage conditions for lyophilized recombinant rat Prolactin?

Lyophilized recombinant rat Prolactin requires careful handling to maintain biological activity:

• Reconstitution should be performed with sterile, buffer-compatible solutions (typically PBS or similar physiological buffers)

• Gentle mixing rather than vortexing prevents protein denaturation

• Short-term storage (1-2 weeks) at 4°C is acceptable for reconstituted protein

• For long-term storage, aliquot and maintain at -80°C to prevent freeze-thaw cycles

When designing experiments, researchers should account for potential activity loss during reconstitution and storage by performing activity validation before critical experiments.

How can researchers effectively purify and characterize prolactin receptors for binding studies?

Based on established methodologies, the most effective purification approach involves:

• Single-step immunoaffinity chromatography using specific monoclonal antibodies to rat liver prolactin receptor

• Scatchard analysis to characterize binding sites (previously demonstrated two classes with Ka = 18.5 x 10⁹ and 1.2 x 10⁹ M⁻¹)

• Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for purity assessment

• Electroelution from gel slices corresponding to Mr 38,000-43,000 for homogeneous preparation

This methodology has shown significant improvements over hormone affinity chromatography, yielding 1000-fold purification with 52% recovery compared to previous approaches (300-fold purification, 15% recovery) .

What analytical techniques are recommended for measuring prolactin-induced prostacyclin production?

For investigating prolactin's vascular effects through prostacyclin production:

• Tissue preparation: Isolate aortic rings and equilibrate in physiologic solution at 37°C

• Treatment: Expose tissue to increasing concentrations of PRL (0.01–10 nmol/L) for defined timeframes

• Sample collection: Remove tissue, freeze supernatant in liquid nitrogen, store at -80°C

• Quantification: Use ELISA with anti-prostacyclin antibody and HRP conjugate

• Normalization: Measure tissue area using stereomicroscopic imaging and ImageJ software

• Data analysis: Calculate concentrations using four-parameter logistic algorithm

This methodology allows precise quantification of PRL-induced prostacyclin release, essential for understanding its vascular effects.

How does recombinant rat Prolactin affect angiogenesis compared to its 16K isoform?

The relationship between full-length Prolactin and its 16K isoform reveals complex, sometimes opposing effects on angiogenesis:

The 16K isoform of rat prolactin, a 16-kDa N-terminal fragment, demonstrates significant anti-angiogenic properties when introduced alongside full-length prolactin in testicular tissue . This antagonistic relationship between the full protein and its fragment provides potential therapeutic applications in conditions characterized by pathological angiogenesis.

What are the differential effects of recombinant rat Prolactin on reproductive parameters?

Prolactin demonstrates complex effects on reproductive parameters:

• Full-length rPRL extends diestrus phase in female rodents

• The 16K rPRL isoform combined with rPRL reduces diestrus duration

• 16K rPRL combined with rPRL unexpectedly increases testosterone levels, while full-length rPRL alone does not significantly alter testosterone production

These findings highlight the importance of considering both the full-length hormone and its proteolytic fragments when investigating reproductive effects, as they can have distinct and sometimes opposing actions on reproductive parameters.

How does recombinant rat Prolactin modulate immune cell function?

Research has established significant immunomodulatory effects of Prolactin:

The 16K isoform combined with full-length Prolactin reduces white blood cell proliferation, whereas full-length Prolactin alone increases proliferation . This demonstrates the complex immunomodulatory role of prolactin and suggests that the balance between full-length hormone and its fragments may be crucial in regulating immune responses in various physiological and pathological conditions.

What experimental approaches are recommended for investigating the antagonistic relationship between full-length Prolactin and its 16K isoform?

To effectively study the antagonistic relationship between these forms:

• Transient gain-of-function animal models using DNA plasmid injection containing either 16K rPRL, rPRL, or both

• Expression validation through RT-PCR, Southern blot, and western blot analyses

• Systematic investigation across multiple biological systems (angiogenesis, immune function, reproduction)

• Quantitative assessment of downstream markers including vessel density, WBC counts, estrous cycle parameters, and hormone levels

This comprehensive approach allows researchers to determine whether 16K rPRL functions as a true antagonist or has independent signaling activities in specific biological contexts.

How can researchers effectively distinguish between direct and indirect effects of recombinant rat Prolactin in complex biological systems?

To differentiate direct from indirect effects:

• Combine in vitro and in vivo approaches to isolate specific cellular responses

• Use receptor antagonists or knockout models to block specific signaling pathways

• Employ time-course studies to identify primary versus secondary effects

• Utilize tissue-specific or inducible expression systems to control Prolactin exposure spatially and temporally

• Perform parallel studies with receptor-binding mutants that maintain structure but lack signaling capacity

These approaches help resolve the complex, pleiotropic effects of Prolactin across different biological systems.

What methodological approaches are recommended for investigating Prolactin's vascular effects?

For investigating vascular tone regulation by Prolactin:

• Tissue preparation: Isolate aortic rings for ex vivo studies

• Experimental conditions: Maintain at 37°C with appropriate oxygenation

• Treatment protocols: Apply increasing concentrations of Prolactin (0.01–10 nmol/L)

• Measurement techniques: Assess both tension changes and biochemical mediators (NO, prostacyclin)

• Data analysis: Use appropriate statistical methods including Student's t-test or one-way ANOVA with Tukey post hoc test

This methodology enables comprehensive assessment of Prolactin's effects on vascular function, including both mechanical responses and biochemical mediator production.

What are common sources of variability in recombinant rat Prolactin functional assays?

Several factors can introduce variability:

Standardization of these parameters is essential for reproducible research with recombinant rat Prolactin.

How should researchers address potential endotoxin contamination when working with recombinant rat Prolactin?

Endotoxin contamination requires systematic management:

• Source selection: Choose preparations with certified low endotoxin levels (<1 EU/μg or >0.05 EU/μg)

• Verification: Independently verify endotoxin levels using LAL assay before critical experiments

• Experimental controls: Include endotoxin-matched controls in experimental design

• Interpretation: Consider potential synergistic effects between Prolactin and low-level endotoxin

• Reporting: Document endotoxin levels in publications to facilitate reproduction

These precautions are particularly important when studying Prolactin's immunomodulatory effects, as endotoxin can significantly confound results.

What strategies help resolve contradictory findings regarding recombinant rat Prolactin's biological effects?

To address contradictory findings:

• Systematically compare protein sources, including expression systems, purification methods, and quality parameters

• Standardize experimental models, considering species, strain, sex, and age variations

• Control for isoform presence, as the 16K fragment may be generated during experimental procedures

• Validate receptor expression and signaling pathway activation in the specific experimental system

• Consider contextual factors including hormone milieu, timing, and physiological state

Contradictory findings often reflect the complex biology of Prolactin rather than experimental error, as its effects are highly context-dependent and may vary across different biological systems and conditions .