Biomedical Engineering Reference
In-Depth Information
4. Now prepare the depletion column by removing the
bottom plug (make sure you keep it, as you need it at the
end of the procedure) and loosen the upper cap. Place the
column in a 2 mL tube. Centrifuge at 2000
g
for 30 s.
5. Remove the screw cap and attach the Luer Lock cap onto
the column. Using a 20 mL syringe filled with Equili-
bration buffer slowly push 8 mL of Equilibration buffer
through the column.
6. Remove the Luer Lock cap, attach the red cap loosely, and
place in a 2 mL tube. Centrifuge at 2000
×
g
for 30 s.
7. Place the column in a fresh 1.5 mL microfuge tube and
add 100
×
L from the diluted, filtered serum. Ensure that
the serum is absorbed into the agarose matrix and not
attached to the plastic of the column. Place the red cap
loosely on top.
8. Incubate for 15-20 min at room temperature.
9. Centrifuge the column at 2000
μ
g
for 30 s. Collect the
flow-through in a 1.5 mL tube. (This is the depleted frac-
tion.)
10. Wash the column by applying 100
×
μ
L Equilibration buffer
and centrifuge immediately at 2000
×
g
for 30 s. Collect this
fraction separately (
see
Note 3
).
11. Repeat Step 10.
12. Attach the Luer Lock and apply 2 mL of 1X Elution buffer
by using a 20 mL syringe. Collect the flow-through (this
is the eluted fraction, containing the high-abundance pro-
teins). Add 0.5 mL of 1 M HEPES solution, pH 7.4, to
adjust the pH every five elution steps.
13. Attach the Equilibration buffer syringe and slowly draw 8
mL of 1X Equilibration buffer through the column.
14. Disassemble the Luer Lock and attach the red cap. Cen-
trifuge at 2000
×
g
for 30 s.
15. Repeat from Step 7 for the desired number of depletions.
It is necessary to undertake 20-30 depletions to obtain
approximately 300-500
g of depleted protein. Always
store the flow-through fractions (depleted and eluted) at
4
◦
C.
16. Concentrate the depleted and eluted fractions using
Microsep Centrifugal devices (at 7500 g in a fixed angel
rotor (34
◦
−
μ
45
◦
) for approximately 8 h at 4
◦
C. Concen-
tration should be ceased when material from 10 depletion
cycles reaches
L in volume (
see
Note 4
).
17. Final depletion step: 100
∼
100
μ
L of concentrated depleted
serum from ten depletions are subjected to a further
μ
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