Clontech Laboratories His60 Ni Superflow Cartridges Benutzerhandbuch - Seite 11

Blättern Sie online oder laden Sie pdf Benutzerhandbuch für Laborausrüstung Clontech Laboratories His60 Ni Superflow Cartridges herunter. Clontech Laboratories His60 Ni Superflow Cartridges 15 Seiten. Purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected cells.

Seite drucken
His60 Ni Superflow Cartridges User Manual
4.
B.

PROTOCOL: Automated Purification on a Liquid Chromatography System

1.
2.
3.
4.
PT5143-1
031716
Lysate clarification
Centrifuge the crude lysate at
supernatant to a clean tube without disturbing the pellet. This is your starting sample.
NOTE: If the supernatant is not clear, centrifuge a second time or filter through a
0.45 µm membrane (e.g., cellulose acetate) to avoid clogging the IMAC column with insoluble
material.
Equilibration
Equilibrate the cartridge and all buffers to
LC System Set up
a. Prepare the LC system by filling the tubing with buffer. On a binary pump LC system, fill
Pump A with
Equilibration Buffer
b. Remove the top plug from the cartridge and start pumping
rate of
until a few drops fill in the top inlet.
1 ml/min
c. Pause the pump, connect the cartridge to the pump outlet, and carefully snap off the bottom
cap of the cartridge (do not twist).
d. Start the pump. To avoid introducing air into the system, allow a few drops to emerge from
the cartridge before connecting to the LC UV monitor inlet port.
Equilibrate Cartridge
Equilibrate the cartridge with
of
for 1 ml cartridge or
1 ml/min
NOTE: For maximum extraction and binding, prepare the sample using our xTractor Buffer
(Section V.A). If you used incompatible reagents (Section I.B.) during the extraction, desalt the
sample on a PD-10 column (Section III.G) before proceeding to Step 4.
Sample Loading
Load the clarified sample onto the cartridge at a flow rate of
Clontech Laboratories, Inc. A Takara Bio Company
for
10,000–12,000 x g
20 min
room temperature
and Pump B with
of the
5–10 column volumes
for 5 ml cartridge.
5 ml/min
www.clontech.com
at 4°C. Carefully transfer the
or
4°C.
Elution
Buffer.
Equilibration Buffer
Equilibration Buffer
and collect fractions.
0.5–1 ml/min
at a flow
at a flow rate
Page 11 of 15