I'm trying to clean up an antibody from a serum sample. For this I have the Melon Gel Purification Kit. Has anyone ever used this one?
So I worked my way through the protocol given by the kit.
2 things I was wondering about:
1.) When I put the gel on the column, I used a 1000µL tip and it looked like it was fine. In the manual they say use a "wide bore or cut tip". Is this point important for the efficiancy?
2.) I put the gel on the column and spin it for 1 min. But the gel comes out of the centrifugation-step in a sloped way, because of the centrifugation of course. Doesn't this make the filter-effect of the gel inefficient? Like the one side is very thick and filters a lot of the serum-parts and the other part of the gel is very thin and filters less serum parts?
regarding 1.
Cutting the tip is just to avoid shear forces that could damage the beads - and damaged beads might reduce the flow rate during chromatography.
regarding 2.
A swing out rotor might help to prevent sloping...
As it's no filter effect, actually the slope is not too bad. You're not working at capacity limits, anyway, as the binding efficiency would be too low then.
Actually, with relatively small serum samples you could chose a batch procedure and mix washed gel with the sample, incubate, and then centrifuge - especially, when there is no swing out rotor available...
Thanks a lot for the advices! I will try to focus on these next time.
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