Instructions For UsePreparation of Fibronectin:
Generally fibronectin should be stored frozen or refrigerated at 1-5 mg/ml. If frozen, thaw at 37o
C. Fibronectin should never be vortexed or treated roughly as it precipitates out of solution easily (visible as a granulated precipitate or as a slimy strand or clumps). Any observed insoluble aggregates are very difficult to resuspend. If precipitation is observed, the solution should initially be warmed to 37o
C with gentle shaking, followed by aseptic filtration via a 5 µm (or smaller) cut off syringe filter.
It is also recommended that fibronectin should not be stored long term in buffer solutions containing Mg2+
as these can contribute to fibronectin precipitation over time. General Coating Procedure (for 96-well plates):
1. Plates: Choice of plates can affect the amount of protein successfully coated. Bio-Rad recommends the use of commercially available high protein binding plates.
2. Add between 1-10 µg/ml of fibronectin in PBS buffer for an overnight (or longer) incubation at 2-8o
C (100 µl/well). The optimum amount of protein to saturate the plate surface should be determined by the user. For ELISA applications some users have coated with carbonate buffer pH 9.0 but this is generally not necessary and can damage the cell binding activity of the fibronectin.
3. Regardless of assay type (cell binding, ELISA etc.), it is necessary to block the remaining protein adherence sites on the plate. Therefore, in a separate step add a blocking protein such as 2-5% BSA in PBS for at least 1 hr at room temperature or overnight at 2-8o
C (200 µl/well). Caution: The BSA solution should be 0.2 µm filtered prior to use to remove excess non-specific binding effects in the assay caused by insoluble BSA clumps. The use of RIA-grade (radio-immunoassay grade, suitable for immunoassays) BSA is recommended.
4. Blocked plates can stored in the refrigerator for several weeks or can be decanted and dried and stored for months in a dessicator. Dessicated material should be rehydrated for 15 min with PBS before use.