Isolation of proteins from SDS-PAGE gels for Ab affinity purification
David Drubin
1. Run an SDS-PAGE gel. To maximize the amount of your protein that you can
resolve, use thick spacers and run a single, wide lane. It is useful to first run a
series of increasing protein amounts on a normal gel to determine the maximum
amount of protein that can be resolved. To determine how much protein you can
load on a thicker gel, multiply the maximum amount of protein resolved on the
thinner gel by the cross section of the thick well divided by the cross section of
the thin well.
2. Stain gel with Coomassie blue and destain. Cut out your protein band. Soak it for
15 min. in several changes of coupling buffer (0.1M NaHCO3 pH 8.3, 0.1% SDS).
3. Wick excess buffer off the gel slice. At this point the gel slice can be stored at –
20°C in a sealed tube.
4. Crush the gel into very fine beads. To do this, first cut the gel into small (1cm.
square) pieces with a razor blade. Place the pieces on a large sheet of parafilm.
Roll a plastic 5ml pipet over the pieces, crushing them into tiny beads (wear
gloves!) If you start with gel pieces that have been wicked off to remove excess
moisture, the gel bits will adhere to the pipet. To remove the bits, simply wash
them off with coupling buffer into a plastic screw cap tube.
5. Tumble the gel bits in about two volumes of coupling buffer overnight at room
temperature to create a gel slurry. Depending on the size of your protein and other
properties, ~50% will elute.
6. Place glass wool in the bottom of a funnel and pour the gel slurry into the funnel.
A blue solution containing your protein should drip through (this process is rather
slow).
7. Because Coomassie blue inhibits coupling to CNBr sepharose, it must be
extracted. Bring your protein solution to 1% in SDS.
8. Add 1/10 volume 2M KCl. Set on ice for 30 min. Your protein will precipitate
with the SDS.