Biomedical Engineering Reference
In-Depth Information
PROTOCOL 12.4 Column Chromatography (Heparin Column
Chromatography)
Equipment and reagents
•
Econo-Pac chromatography columns (Bio-Rad)
•
Heparin-agarose, type I (Sigma)
•
5
M
NaCl
•
1
×
PBS-MK: 1
×
PBS, 1 m
M
MgCl
2
and 2.5 m
M
KCl
•
Pipettes (10 and 25ml)
•
Ultrafree centrifugal filter devices (Millipore: BIOMAX 100 K NMWL membrane
15ml vol)
PBS
•
•
Lactated Ringer's solution
•
Centrifuge.
Method
1 Take a new 20ml Econo-Pac chromatography columns, set up stand and place the
column in a clamp.
2 Add 5 ml slurry
l
of heparin-agarose, type I, into the column to drop the liquid by
gravity flow.
3 When the last drop is left in the columns, place a filter above the heparin,
m
forming
about 2.5 ml bed of heparin column.
4 Pre-equilibrate the heparin column with 20ml of 1
×
PBS-MK by gravity flow.
5 Pre-elute the heparin column with 10ml of 1
×
PBS-MK/1
M
NaCl by gravity flow.
6 Equilibrate the heparin column with 25ml of 1
×
PBS-MK under gravity twice.
7 Load the virus-containing fractions (around 7ml up to 28 ml) from purification by the
iodixanol step gradient into the heparin column.
8 Allow the virus-containing fraction through the heparin column under gravity; discard it
because AAV is reabsorbed onto column.
9 Wash the heparin column with 25ml of 1
×
PBS-MK under gravity twice.
10 Put an Ultrafree centrifugal filter device (BIOMAX 100 K) under the heparin
column.
11 Elute the virus into the BIOMAX 100 K filter with 7 ml of 1
×
PBS-MK/1
M
NaCl.
