NovoNectin® GMP Grade

Fibronectin (rFN-CH296, NovoNectin) is involved in cell attachment, spreading, differentiation, and proliferation. It can greatly improve the infection efficiency of retroviruses on mammalian cells. VLA-4 and VLA-5 on the cell surface bind to the CS-1 site and the cell-binding domain on NovoNectin, respectively, and the retroviral vector binds to the heparin-binding domain, thus promoting the transfection efficiency of retroviruses and lentiviruses on cells. NovoNectin® can be mixed with anti-Human CD3 mAb for coating to enhance T cell amplification.
Recombinant Human NovoNectin is expressed by E. coli, and is produced with raw materials of pharmaceutical applicable level. The host protein residue, nucleic acid residue and common pathogens are strictly controlled, and the production and quality management procedures of the product comply with GMP regulations to ensure the traceability of the production process and all raw materials.

Recommendations
Reconstitution and storage:
（1）Make sure the product properly stored and none expired before using.
（2）Please use water for injection for reconstitution, to a concentration > 100 μg/ml.
（3）Sterile condition without pyrogen is required for reconstitution, re-aliquoting, dilution.
（4）After reconstitution, aliquoted solution vials should be stored at -70°C and minimize freeze-thaw cycles preventing denaturation of protein.
Coating:
（1）Calculate the volume of NovoNectin on the basis of 5 μg/cm2 coating area and dilute the protein solution to 20–100 μg/ml with normal saline. Add the protein solution to the coated vessel to cover the vessel surface , at room temperature for 2 h or at 4 °C overnight. * Add the solution to a 24-well plate at 0.5 ml/well, or a 6-well plate or 35 mm dish at 2 ml/well; do not use preprocessed culture vessels.
Viral transfection:
（1）Add the virus supernatant onto the NovoNectin-coated plate at 125–250 μl/cm2 ; * MOI = 1, virus titer > 1 × 10⁸.
（2）At 32–37 °C for 4–6 h for complete virus adsorption; * In case of a lower virus titer, the plate can be centrifuged at 1000–2000 g at 32 °C for 2 h after the virus is added.
（3）Prepare 0.2–1 × 10⁵ cell suspension; * To this end, cells may be activated 24 h in advance.
（4）Add the cells to the previously processed dish at 0.5–2.5 × 10⁴ cells/cm2; incubate at 37 °C for 2-3 days; * In case of a higher virus titer, the virus may be directly mixed with the cells and the mixture shall be added to the NovoNectin-coated dish for culture at an appropriate density.
（5）The infected cells can be cultured in a conventional way.

Appearance: White porosity
Reconstitution: Totally dissolve in stated time
Visible particle: Conform to ChP
pH value: 5.0–6.0
Bioactivity: >70%, cell adhesion efficiency
Host protein: ≤ 0.005%
Exogenous DNA residue: ≤ 100 pg/mg
Mycoplasma test: Negative
Sterility test: Conform to ChP

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