Gel Isolation for Bioactivity?

Ron Tate rtate at bmb-fs1.biochem.okstate.edu
Fri Nov 3 12:05:15 EST 1995


m9h at nwu.edu (Mustafo Hrundi Jr. III) wrote:
>I would appreciate any protocols for cutting out a protein band from a
>12%, 1.5 mm polyacrylamide gel in order to elute the protein and test it
>for bioactivity.
>
>Any information will be appreciated
>
>Thanks
>
>--
>Mustafo the Man
>"the solitude of the ocean is compromised by the waves wave"

Hello,
I have done something similar to  what you're asking about, 12% 1.5mm PAGE.
I Loaded multiple lanes side by side with sample and one lane with MW markers.  After the run I pulled one of the glass plates off and cut the gel parallel with the lanes so I had one piece of gel with the MW stds. lane and a sample lane and another piece with the rest of the sample lanes. Then leaving both pieces laying together I sliced the piece with samples into 2-3mm slices perpendicular to the lanes being sure to nick the slice locations in the gel piece that had the one sample and the MW markers. I then stained the piece with sample and MW markers and took the slices chopped them into smaller pieces and put them into microfuge tubes with enough buffer to cover them and then used a microfuge pestle to grind them up, set them on ice over night and then put them into the microfuge to pellet the slurry and poured off the super.  The super is then ready to assay.
Another technique that I haven't tried yet but seems good, is to use Amicons Micropure apparatus.  They now have what they call a gel nebulizer.  As I understand it you put your gel piece and buffer into this nebulizer put it into the micropure device and spin it.  The gel is forced through a small pore to make a slurry then the super is driven through a filter and into a Microcon concentrator  So you're suppossed to get extraction, filtration and concentration of your protein all in one step.  I think it was designed for DNA and agarose gels so I'm not sure how well it will work with 12% PAG.

Happy Hunting
Ron Tate OSU BMB





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