Fly Supernatant Protein Prep Protocol

Fly Supernatant Protein Prep Protocol

1.   Raise pH of Supes to 8.0 using NaOH, a milky precipitate will form
2.   Centrifuge supes to remove this precipitate.
3.   Soak YM-10 filter membrane to specs and concentrate sample to ~200mL
4.   Dialyze sample to PBS to remove copper, doing 2 exchanges about 6 hours apart. 10x PBS:2g KCl, 14.4 g Na2HPO4, 2.4g KH2PO4 pH 8.0.
5.   Set up Nickel column apparatus, Use double volume resin solution for final resin volume. (4 mL solution for 2 mL resin)
6.   Let ethanol drip through from solution
7.   Wash resin with 3 column volumes water
8.   Wash resin with 3 column volumes Bind buffer (5mM imidazole)
9.   Add imidazole to sample (final 10mM) add sample and let drip through column
10.   After all of sample binds column wash with 20 column volumes bind buffer
11.   Wash column with 3 or 4 column volumes wash buffer (20mM imidazole)
12.   Elute protein with elute buffer (250mM imidazole), do not disturb resin
13.   Spec each fraction at 280 nm till reading hits baseline again.
14.   Concentrate elutant to 4.5 mL in centripreps (YM-10)
15.   Filter sample through .45uM filter, wash filter with 500uL Biacore buffer (final 5mL)
16.   Run sample over G-200 or S-100 size-exclusion column
17.   Collect samples, pool and concentrate
18.   Run silver stains on all samples containing protein for confirmation.
19.   BIAcore