Preparation of Fibronectin
Note: 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 Mg2+ or Ca2+ because it can precipitate over time.
General Coating Procedure
- Plates: Choice of plates can affect the amount of protein that can be coated. A plastic high binding plate is recommended.
- Add between 1–10 µg/mL of fibronectin in PBS buffer overnight (or longer) in the cold (2–8°C) (100 µL/well). The optimum amount of protein, enough to saturate, needs to be determined depending on the lot of fibronectin and plates. For ELISA some people attach using carbonate buffer pH 9.0 but this is generally not necessary and can damage the cell binding activity of the fibronectin.
- 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 hour at room temperature or overnight at 2–8°C (200 µL/well).
Caution: The BSA solution needs to be filtered to remove excess non-specific sticking in the assay caused by insoluble BSA clumps (RIA-grade BSA is usually recommended). Unfiltered material may look clear but the filters will clog while passing the BSA so it is recommended to use prefilters on top of the filter bed to increase the amount of material that will pass.
- Blocked plates can be stored, as is, in the refrigerator for several weeks or can be decanted and dried and stored for months in a dessicator. Desiccated material should be rehydrated for 15 minutes with PBS before use.
Product News & Releases
- Faster Flow Rates for Sterile Filtration - New Millex® syringe filters with Millipore Express PLUS (PES) membrane
- ReNcell® VM Neural Stem Cell Line Models Parkinson’s Disease - New human disease model shows function of PINK1 gene in neural degeneration
- Millipore Issues New Laboratory Filtration Product Guide
- Millipore Launches Progenitor Cell-Enriched Primary Human Epithelial Cell Cultures
Featured Products
- Fast-Trap Lentivirus Purification and Concentration Kit
- Fast-Trap Adenovirus Purification and Concentration Kit
- EasyCyte Plus Flow Cytometry System
- ECM Cell Culture Optimization Arrays
- ESGRO Complete
- GCTM-5 Antibody, clone GCTM-5
- Guava Viacount Assay
- MilliTrace Constitutive GFP Reporter
- MultiScreenHTS + and MultiScreen Vacuum Manifolds
- ENStem-A hES-derived Human Neural Progenitors