Berglund:ySF1

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yBBP-296 Purification

1. Grow BL21 (STAR) cells to an OD600 between 0.8 and 1.4
2. Induce with 0.1 mM IPTG for about 3 or 4 hours at 18 ºC.
3. Spin down to pellet cells and freeze overnight in -80ºC.
4. Resuspend cells in lysis buffer.
5. Sonicate cells at setting 7 for 30 seconds, 3X.
6. Spin down cell debris with JA-20 rotor at 17,000 rpm, 4ºC, for 30 min.
7. Take supernatant and bind to 3 to 4 mL of GST beads for 15 min, 4 °C.
8. Wash protein bound beads 3X with precission protease cleavage buffer.
9. leave BBP bound beads in about 10 mL of cleavage buffer and add 1 aliquot (30 μL of in house precission protease and let the protease cut off the GST overnight. (I am not sure how many activity units of precission protease is needed. You may need to play with how much to use).
10. The next morning take the beads and spin them down (500g) for about 1 min. Take off the 10 mL superntant. Repeat elution two more times with 10 mL at 4°C. Pool elutions together.
11. Take supernatant and run it through a 0.2 μM filter .
12. Run the supernatant over an cation exchange column (S-column). Before loading onto the column either dialyze into 100 mM NaCl (plus S-column buffer and DTT) or dilute the protein so the NaCl concentration is equal to or lower than 100 mM.
13. Elute with a NaCl gradient between 0.100 M NaCl in buffer A and1 M NaCl in buffer B.
14. Collect fractions and run SDS-PAGE gel to determine which fractions contain BBP-296.
15. Pool desired fractions that contain BBP-296 protein and if needed concentrate the protein. If the protein does not look clean, think about cleaning it up by running it over the sizing column.
16. Dialyze protein into protein buffer.


Buffers
Lysis Buffer
25 mM Tris pH 7.5
300 mM NaCl
0.1% Triton X
1 mM DTT

Cleavage Buffer
50 mM Tris pH 7.0
150 mM NaCl
0.1 % Ttiton X-100
1 mM DTT
1 mM EDTA

Protein Buffer
25 mM Tris pH 7.5
100 mM Na Cl
1 mM EDTA
1 mM DTT

Buffer A (low salt)
10 mM MES pH 6.5
20 mM NaCl
1 mM EDTA
1 mM DTT

Buffer B (high salt)
10 mM MES pH 6.5
250 mM NaCl
1 mM EDTA
1 mM DTT