Biology Reference
In-Depth Information
Wash Buffer-1: 50 mM Tris (pH 8.5), 250 mMNaCl, 5 mMMgCl
2
,50
m
MATP,
1 mM PMSF, 7 mM 2-mercaptoethanol, 20 mM Imidazole, 10% sucrose.
Wash Buffer-2: 50 mM Tris (pH 8.5), 250 mMNaCl, 5 mMMgCl
2
,50
m
MATP,
1 mM PMSF, 7 mM 2-mercaptoethanol, 40 mM Imidazole, 10% sucrose.
Elution buffer: 50 mM Tris (pH 8.5), 250 mM NaCl, 5 mMMgCl
2
,50
m
M ATP,
1 mM PMSF, 7 mM 2-mercaptoethanol, 500 mM Imidazole, 10% sucrose.
Severing Buffer I: 10% sucrose, 20 mM HEPES, 300 mM NaCl, 3 mM MgCl
2
,
5 mM DTT, 50
m
M ATP, pH 7.0.
Coomassie Blue stain and destain.
13.1.1.3.1.2
Reagents
Infected insect cell culture
Nickel beads (Qiagen, 30210, Valencia CA)
Chromatography column (Bio-Rad 0.8
4 cm Poly-Prep Chromatography
Columns, 731-1550, Hercules, CA)
Spin column (Millipore, UFC801024, Billerica, MA)
Funnel
Kimwipes
13.1.1.3.1.3
Equipment
Microfluidizer (Avestin Inc., Ontario, Canada)
Ultracentrifuge
RC-6 Sorvall centrifuge with rotors that can hold 50 and 15 ml conicals
Access to a cold room or a refrigerator to place column
13.1.1.3.2
Detailed procedures
1.
Harvest the cells by centrifuging for 10 min at 4
C at 1000
g
. Save samples of
supernatants to troubleshoot using SDS-PAGE gel.
2.
Gently resuspend the pellet in cold lysis buffer. At this point, all steps should be
done on ice or in a cold room.
3.
In order to homogenize the insect cells and recover the recombinant proteins,
pass the cells through a microfluidizer with nitrogen under 15,000-20,000 PSU
twice. The lysate should be clear after lysis. A high-pressure homogenizer
(Avestin EmulsiFlex-B30) can be used as well. It is important to note that lysing
the cells by sonication does not result in functional protein.
4.
To remove cell debris, the lysed cells are centrifuged for 30 min at 91,500
g
at
4
C.
5.
During the centrifugation of the protein, prepare the Ni
2
þ
-NTA beads for use in
affinity purification. First, dilute with 1 ml of ddH
2
O into 1 ml bed volume of
beads. Centrifuge at 1500
g
for 5 min, remove ddH
2
O from beads, and
resuspend in ddH
2
O. Repeat three times. Resuspend in lysis buffer after the last
centrifugation step.
6.
Discard the pellet of cell debris and use the supernatant for the subsequent
steps. Pass the cell lysate through a funnel with a kimwipe to remove all cell
