Amicon
®
Ultra-4 Centrifugal Filter Devices
2
Suitability
Preliminary recovery and retention studies are suggested to ensure suitability for intended use. See the “How to
Quantify Recoveries” section.
Rinsing Before Use
The ultrafiltration membranes in Amicon
®
Ultra-4 devices contain trace amounts of glycerine. If this material
interferes with analysis, rinse the device with buffer or Milli-Q
®
water before use. If interference continues, rinse with
0.1 N NaOH followed by a second spin of buffer or Milli-Q
®
water.
CAUTION:
Do not allow the membrane in Amicon
®
Ultra filter devices to dry out once wet. If you are not using the
device immediately after rinsing, leave fluid on the membrane until the device is used.
How to Use Amicon
®
Ultra-4 Centrifugal Filter Devices
1. Add up to 4 mL of sample (3.5 mL if using a 23° fixed-angle rotor) to the Amicon
®
Ultra filter device.
2. Place capped filter device into centrifuge rotor (swinging-bucket preferred); counterbalance with a similar device.
3.
When using a swinging-bucket rotor
, spin the device at 4,000 × g maximum for approximately 10–40 minutes.
When using a fixed-angle rotor, orient the device with the membrane panel facing up.
For Amicon
®
Ultra 3K, 10K, 30K, and 50K devices
, spin at 7,500 × g maximum for approximately 10–40
minutes.
For Amicon
®
Ultra 100K devices
,
spin at 5,000 × g maximum for approximately 10–20 minutes.
NOTE:
Refer to Figures 1 and 2, and Tables 2 and 3 for typical spin times.
4. To recover the concentrated solute, insert a pipettor into the bottom of the filter device and withdraw the sample
using a side-to-side sweeping motion to ensure total recovery. The ultrafiltrate can be stored in the centrifuge tube.
NOTE:
For optimal recovery, remove concentrated sample immediately after centrifugation.
Desalting or Diafiltration
Desalting, buffer exchange, or diafiltration are important methods for removing salts or solvents in solutions
containing biomolecules. The removal of salts or the exchange of buffers can be accomplished in the Amicon
®
Ultra-4
device by concentrating the sample, then reconstituting the concentrate to the original sample volume with any
desired solvent. The process of “washing out” can be repeated until the concentration of the contaminating microsolute
has been sufficiently reduced. See example below.
Spin to
concentrate
4 mL of
1 mg/mL
protein in
100 mM
NaCl
50 µL of
80 mg/mL
protein in
100 mM NaCl
100 mM
NaCl
50 µL of
80 mg/mL
protein in
11.1 mM NaCl
11.1 mM
NaCl
Spin to
concentrate
Add 3.95 mL of
10 mM NaCl or
exchange buffer
4 mL of
1 mg/mL
protein in
11.1 mM NaCl
Performance - DNA Concentration
We have determined that the Amicon
®
Ultra-4 30K device provides the best balance between recovery and spin time
for double-stranded DNA for base pairs ranging from 137 to 1,159.
Table 1. Typical Recovery of Nucleotides from Amicon
®
Ultra-4 30K Device
Double-stranded
DNA Base Pair
Size
Spin Time
(min)
Concentrate
Volume (µL)
Recovery (%)
137–1,159
10
50–70
> 85
Spin conditions: Fixed-angle rotor, 5,000 × g, room temperature, 2 mL starting volume.