Amicon
®
Ultra-2 Centrifugal Filter Devices
6
Protein Retention and Concentrate Recovery
The membranes used in Amicon
®
Ultra devices are characterized by a molecular weight cut off (MWCO); that is, their ability to
retain molecules above a specified molecular weight. Solutes with molecular weights close to the MWCO may be only partially
retained. Membrane retention depends on the solute’s molecular size and shape. For most applications, molecular weight is a
convenient parameter to use in assessing retention characteristics. We recommend using a membrane with a MWCO at least two
times smaller than the molecular weight of the protein solute that one intends to concentrate. Refer to Table 4.
Table 4. Typical Retention of Protein Markers
Marker/Concentration
Molecular
Weight
Device
MWCO
% Retention
Swinging Bucket
Spin
Time
(min)
% Retention
Fixed Angle
Spin
Time
(min)
α-Chymotrypsinogen (1 mg/mL)
25,000
3K
99
60
99
60
Cytochrome c (0.25 mg/mL)
12,400
100
100
Vitamin B-12 (0.2 mg/mL)
1,350
6
8
α-Chymotrypsinogen (1 mg/mL)
25,000
10K
99
30
99
20
Cytochrome c (0.25 mg/mL)
12,400
100
100
Vitamin B-12 (0.2 mg/mL)
1,350
10
9
BSA (1 mg/mL)
67,000
30K
100
20
100
15
Ovalbumin (1 mg/mL)
45,000
97
97
Cytochrome c (0.25 mg/mL)
12,400
16
15
BSA (1 mg/mL)
67,000
50K
97
15
100
10
Ovalbumin (1 mg/mL)
45,000
50
60
Cytochrome c (0.25 mg/mL)
12,400
9
17
Thyroglobulin (0.5 mg/mL)
677,000
100K
94
30
94
20
IgG (1 mg/mL)
156,000
95
95
Ovalbumin (1 mg/mL)
45,000
12
13
Spin Conditions: Swinging bucket rotor, 4,000 × g, or 35° fixed angle rotor, 7,500 × g, 2 mL starting volume,
room temperature, n=12.
Factors that determine sample recovery include the nature of the protein solute relative to the device MWCO chosen, starting
concentration, and concentration factor. Table 5 provides typical recoveries for Amicon
®
Ultra-2 devices.
Table 5. Typical Concentrate Recovery
Marker/
Concentration
Device
MWCO
Spin Time
(min)
Concentrate
Volume (µL)
Concentration
Factor (x)
Concentrate
Recovery (%)
Swinging
Bucket
Fixed
Angle
Swinging
Bucket
Fixed
Angle
Swinging
Bucket
Fixed
Angle
Swinging
Bucket
Fixed
Angle
Cytochrome c
(0.25 mg/mL)
3K
60
60
55
45
32
45
97
96
Cytochrome c
(0.25 mg/mL)
10K
30
20
48
39
42
51
98
98
BSA (1 mg/mL)
30K
20
15
43
37
46
57
94
94
BSA (1 mg/mL)
50K
15
10
44
30
47
71
93
87
IgG (1 mg/mL)
100K
30
20
37
32
53
63
88
90
Spin Conditions: Swinging bucket rotor, 4,000 × g, or 35° fixed angle rotor, 7,500 × g, 2 mL starting
volume, room temperature, n=8.
Maximizing Sample Recovery
Low sample recovery in the concentrate may be due to adsorptive losses, over-concentration, or passage of sample through
the membrane.
•
Adsorptive losses depend upon solute concentration, its hydrophobic nature, temperature and time of contact with filter device
surfaces, sample composition, and pH. To minimize losses, remove concentrated samples immediately after centrifugal spin.
•
If starting sample concentration is high, monitor the centrifugation process in order to avoid overconcentration of the sample.
Over-concentration can lead to precipitation and potential sample loss.
•
If the sample appears to be passing through the membrane, choose a lower MWCO Amicon
®
Ultra-2 device.