User:Igor R. Kuznetsov/Notebook/RBL-2H3/2010/03/11
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Fibronectin coating protocols
Hamawy1992: 30µg/ml bovine fibronectin in PBS 2h at 37C, wash 3 times with Pipes
Yasuda1995: 10µg/ml human fibronectin in PBS 2h at 37C, wash twice with PBS and fix with 1% BSA in PBS for 2h
- Dilute Fibronectin to desired concentration using serum-free culture Ca2+, Mg2+-free medium or buffer at pH 7-9. The final solution should be suffi- ciently dilute so that the volume added to the coating surface will coat it evenly (e.g. for a final coating concentration of 5μg/cm2, dilute material to 50 μg/ml and add 1 ml/35 mm dish, 3 ml/60 mm dish, etc.). Note: Because of the CAPS component in the HFN preparation, buffers of media containing Ca2+ and/or Mg2+ added to the HFN may result in the formation of insoluble metal hydroxides. This will not occur if the buffer- ing capacity of the diluent brings the pH to 8.0 or lower.
- Add appropriate amount of diluted Fibronectin to culture surface.
- Incubate at room temperature for one hour.
- Aspirate remaining material.
- Rinse plates carefully with dH2O; avoid scraping bottom surface.
- Plates may be used immediately or may be stored at 2-8°C, damp or air dried, if sterility is maintained.
Preparation of Fibronectin:
Generally fibronectin should be stored in the freezer or refrigerator at 1-5 mg/mL and should never be vortexed or treated roughly because it could crash out of solution and cannot be resuspended. It is also best but not absolute that it should not be stored long in buffers containing Mg++ or Ca++ because it can precipitate over time.
General Coating Procedure:
1. Plates: Choice of plates can affect the amount of protein that can be coated. We recommend Costar type 1 plastic high binding plates or perhaps Nunc maxisorp or polysorp plates or a variety of other high protein binding plates.
2. Add between 1-10µg/ml of fibronectin in PBS buffer overnight (or longer) in the cold (2-8°C) (100µL/well). The optimum amount of protein, enough to saturate needs to be determined depending on the lot of fibronectin and the lot of plates. For ELISA some people attach with carbonate buffer pH 9.0 but this is generally not necessary and can damage the cell binding activity of the fibronectin.
3. No matter what type of assay, 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 2-5% BSA in PBS at least 1-hr room temperature or overnight 2-8°C (200µL/well). Caution: The BSA solution needs to be filtered to remove excess non-specific sitcking in the assay caused by insoluble BSA clumps (RIA-grade BSA is usually very good). Unfiltered material may look clear but it is not, you will find out that the filters will clog while passing the BSA so it is recommended to to use prefilters on top of the filter bed to increase the amount of material that will pass.
4. Blocked plates can stored, as is, 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.
Coated Substrate Preparation: 1. Outline a coating region (5 mm diameter) for coating substrate in the center of each dish with marking pen.
2. Add 20µl of solution containing substrate at a concentration of 10µg/ml to coating region. Incubate 1 hr.
3. Aspirate off liquid, add 20µl of 1% BSA to block for 1 hr.
4. Aspirate off BSA, add 20µl of inhibitor for 1 hr.
5. Dishes are ready for use in flow assay.