Analysis of metalo-beta lactamase enzyme with GS320 column

Record: 42

Category: Column Selection
Product: SB?
Sample: metalo-bata lactamase enzyme

Question
We are trying to purify a metalo-bata lactamase enzyme (Mw=30KDa) and we know that was successfully purified with a GS320 gell filtration column.
We tried a Superdex 75 GF and didn’t work (the protein was eluted together in the first pick -30%CV). We weren’t ble to determine if the protein just aggregates or was acting like a multimer.
1. Can we connect the column to an AKTA system? (the maximum pressure for AKTA is 10MPa)
2. Buffer conditions? the Zn atoms are essential for preserving the activity of the enzyme.
3. loading capacity – can we load mg’s of sample?

Answer
I hope you had a good weekend. Regarding your questions:
1. Can we connect the column to an AKTA system? (the maximum pressure for AKTA is 10MPa)
Unfortunately, it will require an adapter to connect a Shodex column to AKTA system.
2. Buffer conditions? the Zn atoms are essential for preserving the activity of the enzyme.
Zn will not be a problem when using a GS-320.
3. loading capacity – can we load mg’s of sample?

As I mentioned last week, the sample load is depending on the size of analyte and we do not have a specific information on lactamase enzyme. Based on the information we have, it will probably be about 1mg (to keep ideal theoretical number) if you do not mind having broader peaks with decreased theoretical number, you may be able to inject a larger volume, but it needs to be optimized at your site. The preparative column will be able to handle about 6x more loading volume. But I do recommend you to try an analytical column before purchasing a preparative column to make sure that the column suits your analytical purpose.

 
 
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