Recombinant Rat SCF

Recombinant Rat Stem Cell Factor (SCF) is used in research applications to specifically activate the rat c-Kit receptor, supporting studies in hematopoiesis, mast cell biology, and stem cell maintenance where species-specific ligand-receptor interactions are critical.

Key scientific reasons to use recombinant rat SCF:

• Species-specific activity: Rat SCF reliably activates rat c-Kit (CD117), ensuring accurate modeling of rat hematopoietic and immune cell biology. Human SCF does not efficiently activate murine or rat c-Kit, so recombinant rat SCF is necessary for experiments involving rat cells.
• Hematopoietic stem cell support: SCF is essential for the maintenance, survival, and differentiation of hematopoietic stem and progenitor cells, making it indispensable for ex vivo expansion, transplantation, or lineage tracing studies in rat models.
• Mast cell biology: SCF promotes mast cell adhesion, migration, proliferation, and survival, enabling research into allergic responses, inflammation, and immunology in rat systems.
• Spermatogenesis and melanogenesis: SCF is involved in the regulation of spermatogenesis and melanocyte development, supporting studies in reproductive biology and pigmentation.
• Defined, reproducible activity: Recombinant rat SCF is produced with high purity and consistent biological activity, typically measured by dose-dependent proliferation of TF-1 cells, ensuring experimental reproducibility.
• Reduced experimental variability: Using recombinant proteins minimizes batch-to-batch variability and eliminates confounding factors from animal-derived components, improving data reliability.

Typical applications include:

• Ex vivo expansion and maintenance of rat hematopoietic stem/progenitor cells.
• Differentiation assays for mast cells, melanocytes, or germ cells.
• Studies of SCF/c-Kit signaling in rat models of inflammation, allergy, or cancer.
• Cell culture supplementation for rat primary cells or cell lines requiring SCF for survival or proliferation.

Technical notes:

• Recombinant rat SCF is available as a non-glycosylated or glycosylated protein, depending on the expression system, and is typically supplied as a lyophilized powder for reconstitution.
• Biological activity is confirmed by proliferation assays (e.g., TF-1 cell line), with effective concentrations generally below 10 ng/mL.

In summary, recombinant rat SCF is essential for research requiring precise activation of rat c-Kit, supporting robust and reproducible studies in stem cell biology, immunology, and developmental processes specific to rat models.

You can use recombinant rat SCF as a standard for quantification or calibration in your ELISA assays, provided it is compatible with your assay system and properly validated. Recombinant proteins are commonly used as standards in ELISA, including for SCF, as long as they are of high purity and their concentration is accurately determined.

Key considerations and best practices:

• Purity and Form: The recombinant SCF should be highly purified, ideally >95%, and its concentration should be determined by reliable methods such as HPLC or absorbance at 280 nm.
• Standard Curve Preparation: Prepare a standard curve by serially diluting the recombinant SCF in the same buffer or matrix as your samples to minimize matrix effects.
• Validation: Confirm that the recombinant SCF is recognized by the antibodies in your ELISA kit. Some kits are optimized for specific isoforms or post-translational modifications, so cross-reactivity or epitope differences can affect quantification accuracy.
• Spike Recovery: Perform spike-and-recovery experiments by adding known amounts of recombinant SCF to your sample matrix (e.g., serum or plasma) to ensure accurate quantification and to check for matrix interference.
• Lot-to-Lot Variability: Be aware that different lots of recombinant protein may have slight differences in immunoreactivity, which can affect quantification. It is best to calibrate each new lot against a validated standard curve.
• Documentation: Follow the reconstitution and storage instructions specific to your recombinant SCF lot, as stability and solubility can vary.

In summary: Recombinant rat SCF is suitable as a standard for ELISA quantification if it is validated for your assay, prepared at known concentrations, and its performance is confirmed by appropriate controls and recovery experiments.

Recombinant Rat Stem Cell Factor (SCF) has been validated in published research for several key applications, primarily in the context of hematopoietic and mast cell biology:

• Functional Assays: Recombinant rat SCF is widely used in cell-based functional assays to stimulate proliferation, survival, and differentiation of hematopoietic progenitor cells, mast cells, and other c-Kit–expressing cells. For example, it is a standard component in methylcellulose-based colony-forming unit (CFU) assays to assess rat bone marrow progenitor cell growth and differentiation.

• Cell Culture Supplementation: SCF is routinely added to in vitro cultures to support the expansion and maintenance of hematopoietic stem/progenitor cells, mast cells, and other c-Kit–positive populations. It is also used in protocols for optimizing gene transfer and drug toxicity testing in rat hematopoietic models.

• ELISA (Enzyme-Linked Immunosorbent Assay): Recombinant rat SCF is validated as a standard or control in ELISA assays for quantifying SCF levels or for validating antibody specificity.

• Western Blot: Used as a positive control or standard for detecting SCF protein in Western blot analyses.

• Blocking Assays: Applied in blocking experiments to study the functional role of SCF/c-Kit signaling in various cell types.

• Immunohistochemistry (IHC) and Immunoprecipitation: While more commonly associated with SCF antibodies, recombinant SCF can be used for validation and as a control in IHC and immunoprecipitation protocols.

• Mass Spectrometry and SDS-PAGE: Used for protein characterization, purity assessment, and molecular weight determination.

Key biological contexts in which recombinant rat SCF has been validated:

• Hematopoiesis: Essential for in vitro and in vivo studies of hematopoietic stem and progenitor cell proliferation, differentiation, and survival.
• Mast Cell Biology: Induces mast cell survival, proliferation, and mediator release, making it a critical reagent in mast cell research.
• Signal Transduction Studies: Used to activate c-Kit–dependent pathways (e.g., PI3K/AKT, MAPK/ERK, Jak/STAT) in mechanistic studies.
• Drug Toxicity and Gene Transfer: Incorporated into assays evaluating drug effects on hematopoiesis and optimizing gene transfer protocols in rat models.

Summary Table: Applications of Recombinant Rat SCF in Published Research

These applications are supported by both supplier validation data and published research protocols. If you require references to specific peer-reviewed studies, please specify the biological system or assay of interest.

To reconstitute and prepare Recombinant Rat SCF (Stem Cell Factor) protein for cell culture experiments, follow these best-practice steps:

1. Centrifuge the vial
   Briefly centrifuge the lyophilized protein vial (3,000–3,500 rpm, 5 min) to ensure all material is at the bottom before opening.

2. Reconstitution buffer selection

   • For most applications, sterile 10 mM acetic acid is recommended for initial reconstitution, especially for E. coli–derived SCF.
   • Alternatively, sterile phosphate-buffered saline (PBS) or sterile water can be used, depending on the manufacturer’s instructions and downstream application.
   • If the protein is to be stored long-term after reconstitution, include a carrier protein (e.g., 0.1% BSA or HSA) to stabilize and prevent adsorption.

3. Reconstitution procedure

   • Add buffer to achieve a final concentration of 0.1 mg/mL (100 µg/mL) as a standard starting point.
   • Gently pipette up and down to dissolve the protein. Do not vortex, as this can denature the protein.
   • Allow the solution to sit at room temperature for 15–30 minutes with gentle agitation to ensure complete dissolution.
   • If any particulates remain, gently mix for up to 2 hours at room temperature.

4. Aliquoting and storage

   • Divide the reconstituted solution into small aliquots (≥20 µL) to avoid repeated freeze-thaw cycles.
   • Store aliquots at –80°C for long-term storage (up to 12 months), or at 4°C for short-term use (up to 1 week).
   • For storage at –20°C or –80°C, ensure the presence of a carrier protein to maintain stability.

5. Dilution for cell culture

   • Before use, dilute the stock solution into cell culture medium or PBS containing a carrier protein (e.g., BSA, FBS, or HSA) to the desired working concentration.
   • Typical working concentrations for cell culture assays are in the range of 1–100 ng/mL, but optimal concentrations should be determined empirically for your specific assay.

Key technical notes:

• Avoid repeated freeze-thaw cycles to preserve protein activity.
• Do not vortex during reconstitution; use gentle pipetting or swirling.
• Carrier proteins are critical for stability during storage and dilution, especially at low concentrations.
• Check for endotoxin levels if using in sensitive cell types; most recombinant SCF preparations are low endotoxin (<0.1 EU/µg).

Example protocol for 100 µg lyophilized SCF:

1. Centrifuge vial briefly.2. Add 1 mL sterile 10 mM acetic acid (or PBS/water as appropriate) to achieve 0.1 mg/mL.3. Gently pipette to dissolve. Let stand at room temperature for 15–30 min.4. Aliquot into 20–50 µL portions.5. Store at –80°C (with 0.1% BSA if storing >1 week).6. Dilute aliquots into cell culture medium with carrier protein for use.

This protocol ensures maximal recovery, stability, and biological activity of recombinant rat SCF for cell culture applications.