collagen western blotting - (May/20/2007 )

i want to perform western blotting to detect collagen type I in fibroblast cell culture.
i don't know form where to start.

does an extraction method for ELISA works for western blotting.
and do i make total protein concentration measurement at the beginning or after extracting the collagen

any idea about the gel , standards , controls.

thanks

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I also replied to your question about collagen precipitation.
I use 3-8% tris-acetate gels (from invitrogen, Nupage). As a control you could order rat tail collagen type I and for instance type VI for a negative control. You probably get more fragments, the monomers alpha1:alpha 2 2:1, the dimers and the trimers. The trimeric form will not always come on blot, sometimes it's stuck in the gel because of the size.
An other problem is the specificity of the antibodies. Most antibodies will react with all collagen types on a western blot, with native collagen some are really specific.
I've never looked at concentrations, I always just run a gel and see if I have to add more or less protein, it's hard to measure the concentration for collagen.

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