Scatchard analysis - binding assay
Miguel Van Bemmelen
miguelx at pasteur.fr
Wed May 11 05:11:41 EST 1994
In article <9405051925.AA11586 at post.its.mcw.edu> FRED at bmt.mcw.edu ("Frederick Garbrecht") writes:
>I am trying to do a radioligand cell binding assay in which I
>separate the bound and free ligand by centrifugation of the cell
>suspension through an oil phase. I have used a mixture of
>dibutylphthalate and light mineral oil (85:15 ratio), which should
>have a density of about 1.03. Trouble is, some of the tubes have
>inverted phases after centrifugation; i.e. the oil layer is on the
>bottom, instead of on the top. This puzzles me, since it doesn't
>happen in all of the tubes, but only in ones that have fewer numbers
>of cells. Any clues? Anyone have any other suggestions regarding an
>oil phase to use?
I have performed similar experiments with two different models, namely
Dictyostelium cells and isolated renal glomeruli. In both cases, I worked
with a mixture of two different silicon oils: AR20 and AR200 (Wacker Chemie,
In the case of Dictyostelium cells, the mixture usually was AR20:AR200 11:4.
I never had any problem when working with Dicty cells.
However, in the case of glomeruli, I had similar problems to those which you
describe. Either the glomeruli would not get through the oil or they would
start rising slowly through the oil layer after a while. In the first case,
this occurred especially when centrifuging low glomeruli number.
I am not sure which is the reason for this. It can be that at the water-oil
interface, because of the hydrophobic effect, molecules are highly ordered
presenting thus a far stronger barrier to cross than the oil itself.
High cell/glomeruli number will more easily disturb this interface and thus
penetrate the oil layer. Another problem was the temperature at which we
worked. I usually needed to change the composition of the oil mixture,
depending on the working temperature, especially during the centrifugation
step (oil for the summer, oil for the winter... :-) )
In order to avoid cells rising through the oil, freeze the tubes as soon as
possible after centrifugation, at -80C better than at -20C.
I hope this helps. Best regards.
* Michael van Bemmelen, PhD. miguelx at pasteur.fr *
* Unite de Biochimie Cellulaire ph: +33-1-4568-7265 *
* Institut Pasteur fax: +33-1-4568-8399 *
* 28, rue du Docteur Roux *
* 75724 Paris-CEDEX 15 *
* France *
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