Purifying basal lamina from cells

Jarkko Kortesmaa look at signature.below
Tue Dec 8 13:41:36 EST 1998


Julie Wasylnka wrote in message <3666E50F.B25C5C2A at sfu.ca>...
>Does anyone have a protocol for purifying basal lamina from adherent
>tissue culture cells. I would like to extract preferably all the
>proteins from the basal lamina and use them to run over a column. (so
>they must re-fold and be functional).


I'm afraid what you are asking might be quite tricky. The proteins of basal
laminae are difficult to solubilise without using strongly denaturing
conditions. However, some proteins are bound in a calcium-dependent manner
and can be extracted using EDTA. Some components may need partial and/or
selective proteolytic digestion in addition to EDTA to be released. To make
matters worse, the amount of basal lamina material made by cells is usually
rather low.

The pioneering experiments on basal lamina composition were made using
Engelbreth-Holm-Swarm tumor (also known by acronym EHS) -derived material.
This tumor makes huge amounts of (certain) basal lamina components. Maybe
you could look back in the literature and see what people did with these
cells (late 70's and the 80's) and try to adapt some protocols for your use.
One of the pioneers was Rupert Timpl, look after his articles in Medline.

Good luck!

    Jarkko Kortesmaa
    Karolinska Institute, Dept. Matrix Biology
    Sweden






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