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?
>Thanks,
>


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,
Muenchen FRG). 

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

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*       Michael van Bemmelen, PhD.           miguelx at pasteur.fr       *
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