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Pronectin F Plus coating of cell culture supports is suggested whenever optimal cell adhesion is sought.
It is a positively charged, water-soluble variant of the original ProNectin F protein polymer. Pronectin F Plus combines elements of the functionality of fibronectin, collagen, and polylysine with the stability and ease-of-use of ProNectin F.
Instructions for Use
The standard coating kits contain 1 mg or 5 mg of lyophilized and autoclaved Pronectin F Plus per vial.
The coating procedure requires standard tissue culture aseptic techniques. Store the kit contents at room temperature. Keep the stock solution sealed in order to avoid evaporation or contamination.
Start by preparing the stock solution: Add 1 ml/mg of deionized water to vial to dissolve the Pronectin F powder. Swirl until powder is completely dissolved.
This leads to the stock solution concentration of 1 mg/ml ProNectin F Plus.
Coating of cell culture substrates:
Prepare only the amount of coating solution intended for use. Once the stock solution is diluted, ProNectin F will eventually precipitate. Also, the diluted coating solution has a short shelf life and must be used within 48 hours.
Dilute the stock solution to the desired concentration with phosphate buffered saline (PBS) to obtain the coating solution. We recommend performing serial dilutions to determine the optimal coating concentration for your particular cell culture system. Typically, the effective concentration will be 10 μg/ml.
Completely cover the culture surface with the coating solution. Leave for 5 - 10 minutes at room temperature.
Remove the coating solution Rinse substrate twice with PBS.
Seed cells onto the coated substrate immediately or allow substrate to dry for use at a later time. Coated substrates may be stored dry up to two years at room temperature without loss of activity.
GENERAL REMARK: After coating with Pronectin always handle the cell culture support with care, for example pipette very carefully at the edge of the dishes in order not to wash off Pronectin or cells.
Description: ProNectin F Plus is a water-soluble protein polymer which exhibits multiple copies of the RGD cell attachment sequence from human fibronectin and has a net positive charge.
Molecular Weight: Based on its gene sequence, ProNectin F Plus is a protein of 72,738 daltons. As determined by SDS-PAGE, using soluble proteins as molecular weight standards, the approximate apparent molecular weight is 110,000. Differences are due to the unusual amino acid composition of the protein.
Activity: Tested for the promotion of adherence of VERO cells.
Purity: The ProNectin F Plus polymer constitutes ≥ 75% of the total product dry weight as determined by amino acid compositional and microchemical analyses.
Leachable Endotoxins: Leachable endotoxins must be ≤ 0.250 EU/ml for the lot to pass Quality Assurance.
Sterility: The final product is tested according to USP Chapter 71.
Samples tested must show the lot to have no bacterial, fungal, or yeast growth after 14 days.
Stability: Storage tests indicate that the protein powder and coated substrates retain their performance for at least 2 years at room temperature.
How to use best 100 ul of Pronectin F plus
Dilute the sample in 10 ml of Phosphate Buffered Saline (PBS)
Sterile filter (0.22 µ filter type) into a sterile tube
Start coating of cell culture supports (flasks, dishes, microcarriers etc.) using always the minimum volume required to cover the entire cell culture surface. Proceed as described in point 2. of the Instructions for Use sheet.
(Technical tip: you may pool and reuse excess Pronectin to coat immediately additional cell culture substrates – in this way you use only the amount that has been used to coat the previous surface).