Cell lysis and Ni-NTA Affinity Purification

Introduction: The following procedure is for lysing bacterial cells with triton lysis
buffer. For optimal activity, it is suggested that reagents be made right before lysis.
This protocol is also designed for Ni-NTA affinity purification, as the lysis buffer
resembles the equilibration buffer.

Triton lysis buffer:

50mM phosphate buffer (pH 8)
300mM NaCl
10mM imidazole (pH 7.4) (Optional for downstream Ni-NTA affinity purification)
1% triton x-100
Note: The above can be made and stored either in 4C or at room temperature for
about a month (store in the dark if containing imidazole).

Right before cell lysis, add the following:

100ug/mL of lysozyme
One Roche EDTA-free protease inhibitor tablet per 10mL
1uL/mL of 2500U/mL DNase (adjust the solution appears viscous during the lysis).
Note: It is optimal to use the lysis buffer fresh, however this complete lysis buffer
may be usable up to a month if stored in -20C with minimal thawing cycles.

-Lysis protocol:
1. Obtain the frozen cell pellet, keep on ice.
2. To the frozen cell pellet, add lysis buffer to a ratio of 1:10 grams of cell pellet
to mL of lysis buffer.
3. Incubate 5 minutes on ice.
4. Vortex vigorously for 20 seconds, incubate on ice for 5 minutes. A pipette
can be used to carefully re-suspend the pellet if vortexing is insufficient.
5. Repeat Step 4 two to four times, until the pellet is sufficiently broken up.
6. Centrifuge at 4C at max speed for 10 minutes. Retrieve supernatant (lysate).
The pellet can be kept for downstream analysis if desired.
7. Store at -80C or -20C (-80 recommended) until use.
Ni-NTA Affinity purification (modified from the Thermo protocol):
Buffers:
1. Equilibration buffer:
a. 50mM phosphate buffer (pH 8)
b. 300mM NaCl
c. 10mM imidazole (pH 7.4)
2. Wash buffer:
a. 50mM phosphate buffer
b. 300mM NaCl
c. 25mM imidazole
3. Elution buffer:
a. 50mM phosphate buffer
b. 300mM NaCl
c. 250mM imidazole

-Procedure
1. Add one volume of resuspended Ni-resin to a container with 3-4 times the
volume of resin added.
2. Add two resin bed volumes of equilibration buffer, mix by rotating for 5
minutes. Ensure that the resin is fully resuspended at each step.
3. Centrifuge for 2 minutes at 700g. Discard supernatant.
4. If the above lysis buffer was not used, add protein sample to equilibration
buffer at a 1:2 v/v ratio for a total volume greater than the resin bed volume
if (Usually two volumes). For example, for 1mL of resin, add (1mL of protein
sample + 1mL of equilibration buffer). Save at least 20uL of whole cell lysate
for SDS-PAGE analysis.
5. Add protein to resin, rotate for 30 minutes. Keep cold if possible.
6. Centrifuge at 700g for 2 minutes. Remove supernatant, save for analysis.
7. Add two resin-bed volumes of wash buffer. Rotate for 5 minutes. Centrifuge.
Remove supernatant. Save for analysis.
8. Repeat wash 3 to 5 times (more if necessary).
9. Elute in one resin-bed volume of Elution buffer. Rotate for 10 minutes.
Centrifuge 2 min at 700g. Save supernatant for analysis.
10.Repeat Elution 2-4 times.
Note: Keep the sample chilled as often as possible. After running on SDS-PAGE,
adjust the wash steps and elution steps if necessary. Pool any elution fractions with
desired sample.