Pierce Biotechnology
PO Box 117
(815) 968-0747
thermofisher.com
3747 N. Meridian Road
Rockford, lL 61105 USA
(815) 968-7316 fax
2
Procedure for Desalting or Buffer Exchange
Additional Materials Required
•
For 75µL and 0.5mL spin columns: Bench-top microcentrifuge (1500
×
g
) and 1.5mL microcentrifuge tubes
Note:
Use a centrifuge that can be adjusted to 1500
×
g
, such as the Thermo Scientific™ Sorvall™ Legend Micro 17
Microcentrifuge
•
For 2 and 5mL spin columns: Centrifuge (1000
×
g
) and 15mL conical tubes
•
For 10mL spin columns: Centrifuge (1000
×
g
) and 50mL conical tubes
•
For the desalting plates: Variable-speed centrifuge with rotor and carrier capable of handling stacked plates
(height = 4.4cm) at 1000
×
g
•
Wash/equilibration buffer
Note:
Use the same wash/equilibration (stacker) buffer as is desired for the final sample solution. Equilibrating the
desalting resin before sample loading is necessary to ensure proper buffer exchange.
Procedure for Protein Desalting
Note:
See
Table 1
for centrifugation times and volumes for the buffer, stacker and sample for each device. For maximum
protein recovery, add a stacker on top of the applied sample for volumes below the volume specified in
Table 1
.
1.
Remove the column’s bottom closure or the plate’s bottom sealing material. Loosen cap (do not remove cap).
2.
Place the column into a collection tube or plate on top of a wash plate and centrifuge to remove the storage solution.
3.
Discard flow-through and replace the column back into the collection device.
4.
Add wash/equilibration buffer on top of the resin. Centrifuge device and discard flow-through. Repeat this step two
additional times.
Note:
After each spin, the resin should appear white and free of liquid. If liquid is present, make sure you are using the
correct centrifugation speed and time. Incomplete centrifugation may result in poor sample recovery or sample dilution.
5.
Blot the bottom of the column or plate to remove excess liquid. Transfer device to a new collection tube or plate.
6.
Apply sample on top of the resin. If needed, add a stacker as soon as the sample has entered the resin. Adding a stacker is
optional but recommended for dilute protein solutions or small sample volumes to ensure maximum sample recovery.
7.
Centrifuge and retain flow-through that contains sample. Discard spin column or plate.
Table 1. Centrifugation times and volumes for the buffer, stacker and sample.
Column or Plate
75µL
0.5mL
2mL
5mL
10mL
Plate
Sample Volume Range (
µ
L)
2-12
30-130
200-700
500-2000
700-4000
20-100
Wash/equilibration Buffer Volume
50µL
300µL
1mL
2.5mL
5mL
250µL
Sample Volume (µL)*
< 5
< 70
< 350
< 750
< 1500
< 30
Optional Stacker Volume (µL)*
3
15
40
100
200
10
Centrifuge Speed (
×
g)
1000
1500
1000
1000
1000
1000
Centrifugation
Time (min)
Storage Solution
Removal
1
1
2
2
2
2
Wash 1
1
1
2
2
2
2
Wash 2
1
1
2
2
2
2
Wash 3
1
1
2
2
2
2
Sample Recovery
2
2
2
2
2
2
*When using the indicated sample volumes, use a stacker to achieve the highest recovery. The stacker is a volume
of wash/equilibration buffer applied after the added sample has completely entered the desalting resin bed.
