[Protein-analysis] Re: info about gradient maker

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Thu Aug 3 08:00:30 EST 2006


zahid khan wrote:

> Hello
>   Hope u all fine.
>   I want to make my own gradien maker and columns for ionexchange
> chromatography(CM cellulose).
>   Can any one give me some information on the dimentions of the column,
> length and diameter, 
>   similarly the diameter and length of the tubes for gradient maker.I
> want to connect two tubes through a small connecting tube with stopcock.


You need to determine the amount of gel you need first, which depends on
sample size and protein concentration. Once you know this volume, you
can fill the column (for IEC short and thick). For the gradient about 10
to 20 column volumes is appropriate. This depends on the resolution of
your gel. 

So for a 50 ml "fast flow" gel column used for the first purification
step one would not use more than 500 ml gradient, may be even less. For
a column with 1 ml "super fine" gel used for final polishing 20 ml
gradient volume or even more is appropriate. 

Appart from these rules of thumb you want to optimise your procedure to
use the minimal possible gradient volume, to save time and to minimise
protein denaturation on the column. 


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