Hello
I’m working on cell adhesion assay on fibronectin coating. I have prepared aliquote of fibronectin (50µg/ml of steril water) freezed at -20°C. However, when I thawed my aliquot, there was white suspended precipitate (probably fibronectine). I have tried to resuspend my protein but it has not been effective.
Have you ever observed this phenomenon ? If you do, how did you resuspended fibronectin (with PBS) ?
Thanks for your help
The original name for fibronectin (the plasma form, at least) was "cold-insoluble globulin", which should give you some clue. The typical procedure for a 1mg/ml solution is to gently shake at 37C for at least 30min - and you should expect some small amount of insoluble material to remain. Good luck!
The original name for fibronectin (the plasma form, at least) was "cold-insoluble globulin", which should give you some clue. The typical procedure for a 1mg/ml solution is to gently shake at 37C for at least 30min - and you should expect some small amount of insoluble material to remain. Good luck!
I know that commercial fibronectin is stored in 0.05 M Tris-buffered saline, pH 7.5, if that's any help.
First fibronectin should always be stored in a physiological saline solution PBS or TBS. It doesn't matter. Second, I agree with Tristan on all points except shaking the tube. I have worked with fibronectin solutions up to 10mg/ml and my experience is that you have to place the tube in a 37C water bath without agitation for about 30 min. It is the introduction of air bubbles into the solution which causes the precipitation.
Some amount of precipitate will form when thawing fibronectin. We prepare and -80oC freeze Fn in PBS at ~1mg/mL in 1mL aliquots. Thaw rapidly in a 37oC bath wihtout agitation. The precipitate that forms coming out of solution is useless, so fuge it out and recover the soluble supe. Requantify if desired, but the concentration changes very little. Keep the thawed aliquot at 4oC for use; good for several months.
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