To coat a 96-well plate: Full size recombinant protein or rather a 40kd/12kD fragment?
Is a Human Plasma Fibronectin Purified Protein appropriate?
(Millipore cat#FC010, or R&D Systems cat#1918-FN , for example)
Which lymphocyte specifically u want to be get adhered?
Ur choice for human plasma fibronectin purified protein is appropriate.
Suggested Procedure for Coating Cell Cultureware
1. Determine the amount of HFN needed to coat culture vessels by multiplying the total surface
area (cm2) by the desired concentration (µg/mL) of HFN. Recommended amount is 2-10
µg/cm2.
2. Wet the surface of each culture vessel to be coated with a minimum amount of sterile balanced salt solution (serum and protein free) required to cover the entire area.
3. Introduce the proper CO2 atmosphere, if required.
4. Add the calculated amount of HFN to each culture vessel.
5. Allow HFN to adsorb to the surface of the vessel for 5-20 minutes.
6. Remove residual balanced salt solution before proceeding with standard cell culture procedures .
All d best
hi, you can use plasma fibronectin , titre for the concentration of fibronectin at which u get saturation in adhesion. If you invest a day you can isolate fibronectin from human plasma very easily, there is a paper in which detailed protocol is given that how to isolate plasma fibronectin using simply NCP membrane. fibronectin is of good quality and can be used for cell culture work. This will make u free from the constrain of fibronectin availability. For coatin you can use bicarbonate buffer 0.1 M pH 9.6 or PBS pH 7.4 and coat on ELISA plate and keep it overnight at 4oC. it gives best result. next u allow the palte to come to room temp. followed by two washes with sterile PBS and can procced for your experiment and do rest as per your protocol.
A rapid method for fibronectinpurification on nitrocellulosemembranes suitable for tissue culture
Silvia Chifflet, a, , Carmen Bolattob, Susana Tolosaa
a Departamento de Bioquı́mica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay
b Departamento de Histologı́a y Embriologı́a, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800 Montevideo, Uruguay
please this paper it will help u to get purifioed protein easily.
We made good experience also with the 1141 from Sigma. We use frog cells, but tried several fibronectins. Especially with dried powders we got problems several times and switched to the soluble form without any problems since years.
Thank you very much for all your answers!
I was sceptical we could use bovine fibronectin to test adhesion of human lymphocytes, but apparently that's working.
I will most likely test the Fibronectin isolated from human plasma,
best regards
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