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MOLECULAR BIOLOGY: WORKING WITH PROTEINS
PURIFICATION: EXTRACT FROM CELLS
PURIFICATION USING BIOGEL P-10™ SPIN COLUMNS
Purification Using Biogel P-10™ Spin Columns
Bio-Gel P-10 columns are composed of polyacrylamide beads with a molecular weight exclusion limit of 10,000. This allows for high-resolution gel filtration. The gels are prepared by copolymerization of acrylamide and N,N'-methylene-bis-acrylamide. Procedure
1. Place three holes in the bottom of a 0.5 ml microcentrifuge tube using a 25-gauge syringe needle.
2. Fill approximately 2 to 3 mm of the tube with glass beads.
3. Layer BioGel P-10™ slurry on top of the beads and place the 0.5 ml tube inside a 1.5 ml microcentrifuge tube.
4. Centrifuge twice in a clinical centrifuge (or equivalent) at 1,200 X g for 1.5 min (see Hint #1).
5. Apply approximately 50 to 100 μl of the sample to the top of the resin.
6. Centrifuge for 3 min at 1,200 X g in a clinical centrifuge (or equivalent).
7. Collect the supernatant (this constitutes the flow-through) which will contain material above the molecular weight exclusion limit. If the sample is radioactive, the fractionation may be monitored using a geiger counter.
TE 10 mM Tris, pH 8.0
1 mM EDTA
Glass Beads Wash in 1 M HCl
Wash in ddH2O
Sigma 425-500 micron glass beads
Wash in 1 M NaOH
BioRad BioGel P-10™, medium 100-200 mesh Prepare in TE as an 80% slurry BioReagents and Chemicals
BioGel P-10 Spin Columns
1. To centrifuge, place the 1.5 ml microcentrifuge inside a 13 x 100 mm glass tube in the tube adaptor for the centrifuge. Remove the liquid from this 1.5 ml microcentrifuge tube between centrifugation runs.