Andrew Borst
UPDATED ON 4/6/12
Hsp27 FL Purification Protocol
[GROWTH AND EXPRESSION]
1. Grow cells at 37C in 1 L of LB until an OD600 of 0.5-0.7 is reached.
2. Induce cells with 0.5 mM IPTG and let cells express HSP27 for 4 hours.
3. Harvest cells in lysis buffer (15 mL of 50 mM Tris/Hcl, pH 8.0, 100 mM Nacl, 1
mM EDTA).
4. Store cells in -80C freezer until needed.
[PURIFICATION – DAY 1]
1. Thaw cells from freezer and bring total volume of 1 L cell pellet to 30 mL in the
aforementioned lysis buffer.
2. Add 160 uL of saturated PMSF (~43 mg/mL) to resuspended cell pellet.
3. Add 2 mL lysozyme (25 mg/mL in lysis buffer).
a. Incubate on ice for 20 minutes with occasional agitation.
4. Add 80 mg of solid deoxycholate and transfer cells to 37 degree shaker (slow
shake @ ~100-150 rpm in a small Erlenmeyer flask).
a. Let incubate for 15 minutes.
5. Add a small amount of solubilized (in lysis buffer) DNase and RNase to cell
mixture.
a. If you don’t dissolve the DNase and RNase before adding it to the lysed
cell mix, it will not go into solution and you will have to wait an incredibly
long time before you can continue your prep.
6. Add MgCl to 10 mM.
7. Return mixture to 37C shaker and let shake for ~15 minutes (viscosity should
decrease substantially during this time).
8. Centrifuge solution for 15 minutes at 15,000 rpm in SS34 rotor at 4C.
9. Transfer supernatant to room temp and add a saturated solution (3.9 M in
buffer) of ammonium sulfate, pH 7.0 drop-wise under constant stirring until a
final saturation of 40% is achieved.
a. NOTE: Basically, add about to 20 mL to 30 mL lysate (50 mL total volume).
10. Continue stirring for an additional 30 minutes at room temperature.
11. Centrifuge sample for 10 minutes at 15,000 rpm in SS34 rotor at 20C.
12. Discard the supernatant.
13. Redissolve 1 L pellet in 30 mL of “Hsp27 Buffer 1” (20 mM Tris/HCl, pH 7.6, 10
mM MgCl2, 30 mM ammonium chloride, 0.5 mM dithiothreitoil, 0.05 mM NaN3 ,
and some PMSF).
a. Add more PMSF (60 uL).
Andrew Borst
UPDATED ON 4/6/12
14. Run sample over the G-25 column that was pre-equilibrated with “Hsp27
Buffer 1” (20 mM Tris/HCl, pH 7.6, 10 mM MgCl2, 30 mM ammonium chloride,
0.5 mM dithiothreitoil, 0.05 mM NaN3 , and some PMSF).
a. After fractions come off of the column, you will notice a fair amount of
precipitate forming in the fraction tubes containing protein.
15. Centrifuge pooled protein fractions for 10 minutes at 15,000 rpm in SS34 rotor
at 20C.
16. Take supernatant and run sample over an pre-equilibrated DEAE column in
“Hsp27 Buffer 1” via method “ajb Hsp27 FL DEAE voller test”
a. Line A: 20 mM Tris/HCl, pH 7.6, 10 mM MgCl2, 30 mM ammonium
chloride, 0.5 mM dithiothreitoil, 0.05 mM NaN3 , and some PMSF.
b. Line B: 20 mM Tris/HCl, pH 7.6, 10 mM MgCl2, 30 mM ammonium
chloride, 0.5 mM dithiothreitoil, 0.05 mM NaN3 , some PMSF, and 200
Mm NaCl.
i. Hsp27 should elute as a single peak.
17. Pool fractions containing Hsp27. Keep the tailing end of the main peak
separate and label as “tail pool.”
18. Precipitate pooled fractions overnight at 4C in the cold room after adding solid
ammonium sulfate to a final saturation of 50% (1.95 M).
[PURIFICATION – DAY 2]
1. Next morning, centrifuge the precipitated protein for 10 minutes at 15,000
rpm at 20C.
2. Re-dissolve the pellet by hand (shake it in an SS34 tube slowly while walking
around the lab…or something) in 2 mL of “Hsp27 Buffer 1.”
3. Dialyze three times against “Hsp27 Buffer 1” for 6 hours total at 4C (600 mL
each).
4. Centrifuge again for 10 minutes at 15,000 rpm in SS34 rotor at 20C.
5. Supernatant contains Hsp27 and should be stored as aliquots at -80C.
a. Run a 15% SDS-PAGE gel to check purity: _______________
b. Determine protein yield via UV vis: ___________________
[PURIFICATION – MONO Q TEST-RUN]
1. Pre-equilibrate MonoQ column with “Hsp27 Buffer 1” and load your sample onto
the column.
2. Run the Hsp27 method designed by Ying and collect relevant fractions for
analysis.
3. Run an SDS-PAGE gel to determine purity of eluted protein and pool cleanest
fractions.
4. Dialyze into “Hsp27 buffer 1” via the 5 mL HiTrap Deslating column.
5. Determine concentration/amount of protein via UV vis, flash freeze with liquid
nitrogen, and store in 1.5 mL aliquots in the -80C freezer.