Can anybody find me a protocol of coating typeIV collagen? - (Nov/30/2006 )
I have to coat dishes with type IV collagen,but can't find a common protocol,anyone have the experience?
I've been used several substrates. I cover the dish surface with some few ml of Col-IV (25ug/ml) or p-L-Lys (stock) or Gelatin (0.1%), just enough to cover surface. Incubate at RT on culture hood around 3-5 min. Recover the excess of liquid (I re-use it once, some people don't) and let the dish uncover until dry completely. Then the dishes are ready to use.
Thank you for your good suggestions.Do you a protocol for preparation of Collagen IV stock solution? I hearg some one use PBS containing 30%ethanol to dillute the stock solution to coat,but I couldn't cover the surface evenly, the collagen maty become crystalized, I don't know why,hoping to get your experience,thanks again!
I just dissolve Collagen-IV (powder, from Sigma) in PBS 1x. For my stock solution (250 ug/ml) I add 20 ml PBS to 5 mg of Collagen-IV vial, and for use I diluite 1:10 (final 25ug/ml). It works fine.