Millipore Amicon Ultra-2 User manual

Amicon®Ultra-2 Centrifugal Filter Devices 1
User Guide
Amicon®Ultra-2
Centrifugal Filter Devices
for volumes up to 2 mL
For research use only;
not for use in diagnostic procedures.
Introduction
Amicon® Ultra-2 centrifugal lter devices provide fast ultraltration, with the capability for high concentration factors and easy
concentrate recovery from dilute and complex sample matrices. The vertical design and available membrane surface area provide
fast sample processing, high sample recovery (typically greater than 90% of dilute starting solution), and the capability for
50-fold concentration. Typical processing time is 10 to 60 minutes depending on Molecular Weight Cut O (MWCO). Solute
polarization and subsequent fouling of the membrane are minimized by the vertical design, and a physical deadstop in the lter
device prevents spinning to dryness and potential sample loss. Ecient recovery of the concentrated sample (retained species) is
achieved by a convenient reverse spin step after collecting the ltrate. The device can be spun in a swinging bucket or xed angle
rotor. Amicon®Ultra-2 devices are supplied non-sterile and are for single use only.
The Amicon® Ultra-2 product line includes 5 dierent cutos (Molecular Weight Cut O, MWCO). These devices are for
research use only and not for use in diagnostic procedures.
• Amicon® Ultra 3K device — 3,000 MWCO
• Amicon® Ultra 10K device — 10,000 MWCO
• Amicon® Ultra 30K device — 30,000 MWCO
• Amicon® Ultra 50K device — 50,000 MWCO
• Amicon® Ultra 100K device — 100,000 MWCO
Applications
• Concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids (DNA/RNA samples, either
single- or double-stranded), microorganisms, column eluates, and puried samples
• Purication of macromolecular components found in tissue culture extracts and cell lysates, removal of primer, linkers, or
molecular labels from a reaction mix, and protein removal prior to HPLC
• Desalting, buer exchange, or dialtration
Materials Supplied
10K
4
3
2
1
00
20
00
00
00
Filter Device
Concentrate
collection
tube
Filtrate
collection
tube
The Amicon® Ultra-2 device is supplied with two tubes. During
operation, one tube is used to collect ltrate; the other to cap
the device during concentration and subsequently to recover the
concentrated sample.

Amicon®Ultra-2 Centrifugal Filter Devices 2
Required Equipment
Centrifuge with swinging bucket or xed angle rotor with wells/carriers that can accommodate 17 mm × 100 mm tubes (same
well/carrier size as for Amicon®Ultra-4 devices and the former Centricon® device).
CAUTION: To avoid damage to the device during centrifugation, make sure it is properly assembled and seated at the bottom of
the rotor. The rim of the concentrate collection tube should be inside the rotor well. Check clearance before spinning.
Suitability
Preliminary recovery and retention studies are suggested to ensure suitability for intended use. See the “How to Quantify
Recoveries” section.
Device Storage
Store at room temperature.
Prerinsing
The ultraltration membranes in Amicon® Ultra-2 devices contain trace amounts of glycerine. If this material interferes with
analysis, pre-rinse the device with buer or Milli-Q® water. If interference continues, rinse with 0.1 N NaOH followed by a second
spin of buer or Milli-Q® water.
CAUTION: Do not allow the membrane in Amicon® Ultra lter devices to dry out once wet. If you are not using the device
immediately after pre-rinsing, leave uid on the membrane until the device is used.
How to Use Amicon®Ultra-2 Centrifugal Filter Devices
1. Insert the Amicon® Ultra-2 device into the ltrate collection tube, making sure that the device is fully seated in the tube.
2. Add up to 2 mL of sample to the device and cover with concentrate collection tube. Push the tube rmly onto the device.
WARNING: Failure to fully seat the device in the ltrate collection tube and push the concentrate collection tube rmly onto
the device may result in the device breaking during centrifugation. See gure below.
10K
4
3
2
00
00
00
10K
4
3
2
1
00
20
00
00
00
Make sure both tubes are fully seated onto device
Add sample
4
3
2
1
00
20
00
00
00
10K
3. Place lter device into the centrifuge rotor with one membrane panel facing the center of the rotor (one panel facing up and the
other panel facing down). Make sure the device is seated on the bottom of the rotor well and that the rim of the concentrate
collection tube is completely inside the well. See gures below. Counterbalance with a similar device.
10K
4
3
2
1
00
20
00
00
00
Spin to
concentrate
10K
4
3
00
00
White membrane
panel facing
center of rotor
Orient device correctly in rotor Seat device fully in well
Rim of tube
Concentrate tube
should be inside
4. Spin for approximately 10–60 minutes depending on the MWCO of the device used:
4,000 × g maximum when using a swinging bucket rotor
7,500 × g maximum when using a xed angle rotor
NOTE: When spinning viscous solutions such as undiluted serum or plasma, do not exceed 5,400 x g.
Refer to Figures 1 and 2 and Tables 2 and 3 for typical spin times.
5. Remove the assembled device from the centrifuge and separate the Amicon® Ultra lter device from the ltrate collection tube.
6. To recover the concentrated solute, invert the Amicon® Ultra lter device and concentrate collection tube. Place in centrifuge
and counterbalance with a similar device. Spin for 2 minutes at 1,000 × g to transfer the concentrated sample from the device
to the tube.
NOTE: For optimal recovery, perform the reverse spin immediately.

Amicon®Ultra-2 Centrifugal Filter Devices 3
10K
4
3
2
1
00
20
00
00
00
4
3
2
1
00
20
00
00
00
10K
Invert device and
concentrate tube
10K
4
3
2
1
00
20
00
00
00
Separate device
from ltrate tube
Filtrate Concentrate
Spin to
recover
Desalting or Dialtration
Desalting, buer exchange, or dialtration are important methods for removing salts or solvents in solutions containing
biomolecules. The removal of salts or the exchange of buers can be accomplished in the Amicon® Ultra-2 device by concentrating
the sample, discarding the ltrate, 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 suciently reduced.
See example below.
Spin to
concentrate
2 mL of
1 mg/mL
protein in
100 mM NaCl
50 µL of
40 mg/mL
protein in
100 mM NaCl
100 mM NaCl
50 µL of
40 mg/mL
protein in
12.3 mM NaCl
12.3 mM NaCl
Add 1.95 mL of
10 mM NaCl or
exchange buer
2 mL of
1 mg/mL
protein in
12.3 mM NaCl
10K
4
3
2
1
00
20
00
00
00
10K
4
3
2
1
00
20
00
00
00
10K
4
3
2
1
00
20
00
00
00 10K
4
3
2
1
00
20
00
00
00
Spin to
concentrate
Performance - DNA Concentration
The Amicon® Ultra-2 30K device provides the best balance between PCR recovery and PCR primer removal for double-stranded
DNA for base pairs ranging from 137 to 1159.
Table 1. Typical Recovery of Nucleotides from the Amicon®Ultra-2 30K Device
Swinging Bucket Rotor 35° Fixed Angle Rotor
4,000 × g for 40 min 7,500 × g, for 15 min
PCR
Product
(base
pairs)
PCR
Primer
(bases)
PCR
Recovery
(%)
PCR
Primer
Removal
(%)
Final
Voume
(µL)
PCR
Recovery
(%)
PCR
Primer
Removal
(%)
Final
Volume
(µL)
137
10
20
48
83
87
86
92
80
61
44
43
41
78
75
78
93
86
67
27
22
25
1159
10
20
48
96
97
97
98
93
82
35
39
37
95
93
95
98
93
82
26
26
27
100 µL PCR diluted to 2,000 µL starting volume, n=6

Amicon®Ultra-2 Centrifugal Filter Devices 4
Performance - Protein Concentration
Flow Rate
Factors aecting ow rate include sample concentration, starting vol ume, chemical nature of solute, relative centrifugal force,
centrifuge rotor angle, membrane type, and temperature. Figures 1 and 2 and Tables 2 and 3 can be used to estimate the time
required to achieve a given volume of ltrate or concentrate for a variety of protein markers. A typical spin time for a 2 mL sample
in a xed angle rotor is approximately 10 to 60 minutes (depending on device nominal molecular weight limit). While most of
the sample is ltered in the rst 10 to 20 minutes of centrifugation, the lowest concentrate volume (30–70 μL) is reached after
spinning for 10 to 60 minutes.
Figure 1. Typical Filtrate Volume vs. Spin Time for Amicon®Ultra-2 Device, Swinging Bucket Rotor
0
0.5
1
1.5
2
0 10 20 30 40 50 60
3K
10K
30K
50K
100K
Filtrate Volume (mL)
Spin time (min)
0
0.5
1
1.5
2
0 10 20 30 40 50 60
3K
10K
30K
50K
100K
Spin time (min)
Filtrate Volume (mL)
Spin conditions: Swinging bucket rotor, 4,000 × g, room temperature, 2 mL starting volume.
Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.
Figure 2. Typical Filtrate Volume vs. Spin Time for Amicon®Ultra-2 Device, Fixed Angle Rotor
Spin conditions: 35° xed angle rotor, 7,500 × g, room temperature, 2 mL starting volume.
Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.

Amicon®Ultra-2 Centrifugal Filter Devices 5
Table 2. Typical Concentrate Volume / Concentration Factor vs. Spin Time, Swinging Bucket Rotor
3K device 10K device 30K device 50K device 100K device
Spin Time
(min)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
5 281 7 91 22 1070 2
10 880 2190 11 71 27 47 42 523 4
15 96 21 52 38 44 47 167 12
20 317 7 65 31 43 46 38 52 65 31
30 147 30 48 42 39 51 38 53 37 53
40 102 20 44 45
60 55 32
Spin conditions: Swinging bucket rotor, 4,000 × g, room temperature, 2 mL starting volume.
Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.
Shaded volumes were used for the calculation of protein recovery in Table 5.
Table 3. Typical Concentrate Volume/Concentration Factor v. Spin Time, Fixed Angle Rotor
3K device 10K device 30K device 50K device 100K device
Spin
Time
(min)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
Conc.
Volume
(μL)
Conc.
Factor
(x)
5137 15 80 25 879 2
10 731 3101 21 51 39 30 71 203 10
15 60 33 37 57 22 90 61 34
20 215 10 39 51 24 85 21 99 32 63
30 106 19 25 80 20 101 18 89 17 115
40 70 29 23 87
60 45 45
Spin conditions: 35° fixed angle rotor, 7,500 × g, room temperature, 2 mL starting volume.
Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.
Shaded volumes were used for the calculation of protein recovery in Table 5.