Clontech Laboratories His60 Ni Superflow Cartridges Benutzerhandbuch - Seite 12

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.

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His60 Ni Superflow Cartridges User Manual
5.
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C. PROTOCOL: Manual Purification Using a Syringe
1.
2.
PT5143-1
031716
Wash Step
Wash the cartridge using a flow rate of
cartridges with
8 column volumes
(i.e., Equilibration Buffer containing 40 mM imidazole). The Wash Buffer is
Wash Buffer
prepared on the LC system by running Pump B at 7.1%.
Elution Step
Elute using a flow rate of
approximately
5–8 column volumes
collect
1 ml
fractions.
Monitor protein elution by measuring the absorbance of the fractions at 280 nm or performing a
Bradford assay (Bradford, 1976). The collected fractions can be analyzed by SDS-PAGE.
NOTE: If necessary for downstream applications, remove excess imidazole by gel filtration on a
PD-10 column (Section III.G).
Cartridge Regeneration
Wash cartridge with
20 column volumes
of
volumes
20 mM MES, 0.3 M NaCl pH 5.0
NOTE: Regeneration allows the cartridge to be reused to purify the same protein, without
significant loss of binding capacity, up to 5 times depending on the purification conditions and
the target protein.
Extended Storage (over 1 week)
Wash the cartridge with
4°C Attach supplied bottom cap, followed by the top plug.
Equilibration
Equilibrate the cartridge and all buffers to
Luer Lock Syringe Setup
Fill syringe with
5–10 column volumes
Clontech Laboratories, Inc. A Takara Bio Company
1 ml/min
of
Equilibration Buffer
for 1 ml cartridges or
1 ml/min
of
Elution Buffer
of
Equilibration Buffer
buffer.
of
five column volumes
room temperature
of
Equilibration
www.clontech.com
for 1 ml cartridges or
5 ml/min
followed by
for 5 ml cartridges with
5 ml/min
(containing 300 mM imidazole) and
or wash with
after each use. Store in
water
or at 4°C.
Buffer.
for 5 ml
of
7 column volumes
10 column
at
20% ethanol
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