Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family that is also known as chemokine (C-X-C motif) ligand 4 (CXCL4). This chemokine is released from alpha-granules of activated platelets during platelet aggregation, and promotes blood coagulation by moderating the effects of heparin-like molecules. Due to these roles, it is predicted to play a role in wound repair and inflammation.1 PF4 is usually found in a complex with proteoglycan. PF4 is chemotactic for neutrophils, fibroblasts and monocytes, and interacts with a splice variant of the chemokine receptor CXCR3, known as CXCR3B.2 Platelet factor-4 is released from the alpha-granules of activated platelets and binds with high affinity to heparin. Its major physiologic role appears to be neutralization of heparin-like molecules on the endothelial surface of blood vessels, thereby inhibiting local antithrombin III activity and promoting coagulation. As a strong chemoattractant for neutrophils and fibroblasts, PF4 probably has a role in inflammation and wound repair.3 PF4 is the antigen in heparin-induced thrombocytopenia, an idiosyncratic autoimmune reaction to the administration of the anticoagulant heparin.4
Protein Details
Purity
>97% by SDS-PAGE and analyzed by silver stain.
Endotoxin Level
<0.01EU/µg as determined by the LAL method
Biological Activity
The biological activity of Human PF-4 was determined by its ability to inhibit human FGF-basic-dependent proliferation of NR6R-3T3 fibroblasts (Rizzino, A., et al., 1988, Cancer Res. 48:4266; Thomas, K. et al., 1987, Methods Enzymol. 147:120; Watson, J.B. et al., 1994, J. Clin. Invest. 94:261). The expected ED<sub>50</sub> for this effect is typically 5 - 15 μg/ml.
The predicted molecular weight of Recombinant Human PF4 is Mr 7.8 kDa.
Predicted Molecular Mass
7.8
Formulation
This recombinant protein was lyophilized from a 0.2 μm filtered solution in 25% acetonitrile (CH3CN) and 0.1% trifluoroacetic acid (TFA).
Storage and Stability
This lyophilized protein is stable for six to twelve months when stored desiccated at -20°C to -70°C. After aseptic reconstitution, this protein may be stored at 2°C to 8°C for one month or at -20°C to -70°C in a manual defrost freezer. Avoid Repeated Freeze Thaw Cycles. See Product Insert for exact lot specific storage instructions.
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Recombinant Human PF-4 (Platelet Factor 4, CXCL4) is widely used in research due to its well-characterized roles in immune modulation, coagulation, cell migration, and disease modeling. It offers a consistent, scalable, and biologically active source for diverse experimental applications, enabling reproducible and mechanistic studies.
Key scientific applications and advantages:
Immunology and Inflammation: PF-4 regulates immune cell migration, particularly T cells and neutrophils, and modulates inflammatory responses. It is used to study chemotaxis, immune cell activation, and cytokine signaling in vitro and in vivo.
Coagulation and Hemostasis: PF-4 interacts with heparin and glycosaminoglycans, inhibiting heparin’s anticoagulant activity and promoting clot formation. Recombinant PF-4 is essential for mechanistic studies of coagulation, heparin-induced thrombocytopenia (HIT), and related diagnostic assays.
Antibody Development and Validation: Highly pure, tag-free recombinant PF-4 serves as an immunogen for generating specific antibodies and as a positive control in ELISA, Western blot, and immunofluorescence assays. Its native-like structure ensures reliable antibody recognition.
Cell Migration and Wound Healing: PF-4’s chemotactic activity on fibroblasts and other cell types makes it valuable for wound healing and migration assays, such as scratch assays and transwell migration chambers.
Protein-Protein Interaction Studies: Recombinant PF-4 is used in biochemical assays (e.g., pull-down, surface plasmon resonance, co-immunoprecipitation) to investigate interactions with heparin, glycosaminoglycans, and cell surface receptors.
Neuroscience and Aging: Recent studies show PF-4 can rejuvenate brain function, improve cognition, and attenuate neuroinflammation in aging models, making it a tool for neurodegeneration and aging research.
Disease Modeling and Bioassays: PF-4 is implicated in viral propagation, cancer biology, and autoimmune conditions. Recombinant protein enables controlled studies of its effects on cell signaling, proliferation, and disease mechanisms.
Technical advantages of recombinant PF-4:
Consistency and Scalability: Recombinant production ensures batch-to-batch consistency, high purity (>97%), and scalability for large experiments.
Defined Activity: Biological activity is confirmed, supporting dose-response and mechanistic studies.
Structural Fidelity: Recombinant PF-4 retains native secondary and quaternary structures, including tetramerization and heparin-binding properties, critical for functional assays.
Summary of best practices:
Use recombinant PF-4 for mechanistic studies requiring precise control over protein concentration and purity.
Employ it in antibody development, cell migration, and protein interaction assays where native-like structure and activity are essential.
Select recombinant PF-4 for disease modeling and diagnostic assay development to ensure reproducibility and reliability.
In conclusion, recombinant human PF-4 is a versatile and essential reagent for research in immunology, hematology, cell biology, neuroscience, and translational medicine, providing a reliable platform for both basic and applied scientific investigations.
Yes, recombinant human PF-4 can be used as a standard for quantification or calibration in ELISA assays, provided the assay is validated to recognize recombinant PF-4 and the standard is of sufficient purity and accurately quantified. Several commercial ELISA kits specifically use recombinant human PF-4 as their standard and have demonstrated accurate quantification of both recombinant and native PF-4.
Key considerations:
Assay Compatibility: Ensure your ELISA is validated to detect recombinant PF-4. Many sandwich ELISA kits for PF-4 are designed to recognize both native and recombinant forms, and their standard curves are generated using recombinant PF-4.
Standard Preparation: The recombinant PF-4 standard should be well-characterized, with a known concentration and purity. It should be prepared in the same buffer or matrix as your samples to minimize matrix effects.
Parallelism: Ideally, the standard curve generated with recombinant PF-4 should be parallel to the response obtained from native PF-4 in your biological samples. This ensures accurate interpolation of sample concentrations.
Validation: If you are developing your own assay or using a kit not specifically validated for recombinant PF-4, perform recovery and linearity experiments by spiking recombinant PF-4 into your sample matrix to confirm accurate quantification.
Limitations and best practices:
Some ELISA kits are designed only for native PF-4 and may not be validated for recombinant standards. Always check the kit documentation for compatibility.
Recombinant proteins used as standards should be tested for stability and consistency across batches.
Avoid mixing standards or reagents from different kits or vendors, as this can affect assay performance.
Summary Table: Recombinant PF-4 as ELISA Standard
Kit/Protocol Recognizes Recombinant PF-4?
Standard Curve Validated for Recombinant PF-4?
Notes
Yes (most sandwich ELISA kits)
Yes
Parallelism with native PF-4 demonstrated
No (some competitive ELISA kits)
No
Designed for native PF-4 only
In conclusion: You can use recombinant human PF-4 as a standard for ELISA quantification if your assay is validated for this purpose and you follow best practices for standard preparation and assay validation.
Recombinant Human PF-4 (CXCL4) has been validated for a wide range of applications in published research, including:
Bioassays: Used to study the effects of PF-4 on cell proliferation, migration, and signaling in various cell types, including hematopoietic stem cells, endothelial cells, and cancer cells.
Binding Assays: Employed to investigate interactions between PF-4 and other molecules, such as heparin, viruses, and antibodies.
Functional Studies: Applied in studies assessing the role of PF-4 in processes like heparin neutralization, modulation of immune responses, and regulation of hematopoietic stem cell aging.
ELISA Standards: Used as a standard in ELISA assays to quantify PF-4 levels in biological samples.
Cell Culture: Utilized in cell and tissue culture experiments to examine the effects of PF-4 on cell behavior and function.
In Vivo Studies: Administered in animal models to evaluate the impact of PF-4 on aging, inflammation, and disease progression.
Diagnostic Testing: Used in the development and validation of diagnostic assays, such as the PF4-dependent enhanced serotonin release assay (PF4-SRA) for heparin-induced thrombocytopenia.
Structural Studies: Employed in crystallographic and biophysical studies to determine the structure and function of PF-4 and its interactions with other proteins.
These applications highlight the versatility and importance of recombinant human PF-4 in both basic and applied research.
To reconstitute and prepare Recombinant Human PF-4 (Platelet Factor 4, CXCL4) protein for cell culture experiments, dissolve the lyophilized protein in sterile water or buffer to a concentration of 0.1–1.0 mg/mL, then dilute to your working concentration in cell culture medium containing a carrier protein such as 0.1% BSA or 10% FBS.
Step-by-step protocol:
Centrifuge the vial briefly before opening to ensure all lyophilized material is at the bottom.
Add sterile water or buffer (e.g., PBS or cell culture medium) to the vial to achieve a stock concentration of 0.1–1.0 mg/mL. For example, add 200 μL sterile water to 20 μg PF-4 for a 0.1 mg/mL solution.
Gently mix by swirling or tapping. Avoid vigorous shaking or vortexing, which can denature the protein.
Allow the protein to dissolve at room temperature for 15–30 minutes with gentle agitation.
Aliquot the stock solution to avoid repeated freeze-thaw cycles. Store aliquots at –20 °C to –80 °C for long-term storage, or at 2–8 °C for up to 1 month after reconstitution.
For cell culture use, dilute the stock solution into your culture medium to the desired final concentration (e.g., 1–10 ng/mL for chemotaxis assays). Include a carrier protein such as 0.1% BSA or 10% FBS to stabilize PF-4 and minimize adsorption to plastic.7
References & Citations
1. Poncz, M. et al. (1990) Blood76: 336 2. Romagnani, P. et al. (2003) J Exp Med.197: 1537 3. Warkentin, TE. et al. (2007) N. Engl. J. Med.356: 891
Products are for research use only. Not for use in diagnostic or therapeutic procedures.
