Clontech Laboratories His60 Ni Superflow Cartridges Руководство пользователя - Страница 12
Просмотреть онлайн или скачать pdf Руководство пользователя для Лабораторное оборудование Clontech Laboratories His60 Ni Superflow Cartridges. Clontech Laboratories His60 Ni Superflow Cartridges 15 страниц. Purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected cells.
Печатать страницуHis60 Ni Superflow Cartridges User Manual
5.
6.
7.
8.
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
Page 12 of 15