Ion-exchange Chromatography with Enzymes

[Wolfgang Schechinger]

Rod <sciteach at droog.mbt.washington.edu> wrote:
>We're working in a summer program for Middle School Teachers, trying to 
>develop some labs to use with kids in the 11-14 age range.  One method 
>we're trying is using an ion exchange column to separate two enzymes in 
>solution and show their presence in collected fractions by showing their 
>activity.  We've been using alkaline phospatase (bovine intestinal 
>mucosa) as a sample, but aren't having much luck in getting it to bind 
>to the matrix-- (Bio-Rex 70 - cation) and DE-52 (anion) are two that we 
>have on hand.  We've been testing for AP with pNPP at this point. 
>
>We've tried a ph range from 4-8.  Is there a way to find the optimum pH 
>for a particular enzyme/resin combination?


I would like to recommend a bulletin by Pharmacia in that you will find many procedures on how 
to optimize your problem step by step:

"Ion Exchange Chromatography, Principles and Methods (Part-No. 18-1022-19)
Pharmacia LKB Biotechnology Inc.
800 Centennial Avenue
PISCATAWAY N.J.08855-1327
Phone (908) 457 8000
Fax   (908) 457 0557

>
> Ideally we'd like to find two enzymes from some natural source--like 
>banana or potato extract, or saliva, or beef liver--maybe 
>peroxidase,amylase, catalase or AP that would would show up with a color 
>indicator that is relatively safe to use in the classroom.   We've   
>been using "fast red" as and indicator for AP in some labs, but any 
>enzyme/source/indicator combinations that you know might help us.  Or 
>where else would you send us to ask?
>
>Thanks in advance for your help,
>
>Rod Margason
>Integrated Science Partners 
>
>
>
I hope this helps

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Wolfgang Schechinger
Institute for Diabetes Research
Koelner Platz 1
80804 Munich
Germany
Phone +49 (89) 30 79 31 24
Fax   +49 (89) 30 81 733
email u7k0201 at sunmail.lrz-muenchen.de
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