Amicon
®
Ultra-4 Centrifugal Filter Devices
4
Table 4. Typical Retention of Protein Markers
Marker/Concentration
Molecular
Weight
Device
MWCO
% Retention
Swinging-bucket
% Retention
Fixed-angle
Spin Time (min)
α-Chymotrypsinogen (1 mg/mL)
25,000
3K
> 95
> 95
40
Cytochrome c (0.25 mg/mL)
12,400
> 95
> 95
40
Vitamin B-12 (0.2 mg/mL)
1,350
< 35
< 35
40
α-Chymotrypsinogen (1 mg/mL)
25,000
10K
> 95
> 95
15
Cytochrome c (0.25 mg/mL)
12,400
> 95
> 95
15
Vitamin B-12 (0.2 mg/mL)
1,350
< 15
< 15
15
BSA (1 mg/mL)
67,000
30K
> 95
> 95
10
Ovalbumin (1 mg/mL)
45,000
> 90
> 90
10
Cytochrome c (0.25 mg/mL)
12,400
< 20
< 20
10
Vitamin B-12 (0.2 mg/mL)
1,350
< 10
< 10
10
BSA (1 mg/mL)
67,000
50K
> 95
> 95
10 (SB), 5 (FA)
Ovalbumin (1 mg/mL)
45,000
~ 60
~ 65
10 (SB), 5 (FA)
Cytochrome c (0.25 mg/mL)
12,400
< 10
< 10
10 (SB), 5 (FA)
Thyroglobulin (0.5 mg/mL)
677,000
100K
> 95
> 95
15
IgG (1 mg/mL)
156,000
> 90
> 90
15
Ovalbumin (1 mg/mL)
45,000
< 25
< 20
15
Spin Conditions: Swinging-bucket (SB) rotor, 4,000 × g, or 35° fixed-angle (FA) rotor, 7,500 × g for 3K, 10K, 30K, and 50K,
5,000 × g for 100K, 4 mL starting volume, room temperature, n=6 (mean value of 3 device lots).
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-4 devices.
Table 5. Typical Concentrate Recovery
Concentrate
Volume (μL)
Concentration
Factor (X)
Concentrate
Recovery (%)
Marker/
Concentration
Device
MWCO
Spin
Time
(min)
Swinging-
bucket
Fixed-
angle
Swinging-
bucket
Fixed-
angle
Swinging-
bucket
Fixed-
angle
Cytochrome c
(0.25 mg/mL)
3K
40
94
62
43.5
65.0
98.2
96.7
Cytochrome c
(0.25 mg/mL)
10K
15
76
54
52.3
76.6
97.3
98.5
BSA (1 mg/mL)
30K
10
73
42
56.1
98.6
95.8
95.0
BSA (1 mg/mL)
50K
10
32
23
137.0
177.4
98.8
92.8
IgG (1 mg/mL)
100K 15 (SB),
10 (FA)
36
53
115.9
56.8
92.2
91.3
Spin Conditions: Swinging-bucket (SB) rotor, 4,000 × g, or 35° fixed-angle (FA) rotor, 7,500 × g for 3K, 10K, 30K, and 50K, 5,000 × g
for 100K, 4 mL starting volume, room temperature, n=6 (mean value of 3 device lots). The shaded volumes were taken from Tables 2 and 3.
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 the starting sample concentration is high, monitor the centrifugation process in order to avoid over-concentration
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-4 device.
How to Quantify Recoveries
Calculate total recovery, percent concentrate recovery, and percent filtrate recovery using the method below. The
procedure provides a close approximation of recoveries for solutions having concentrations up to roughly 20 mg/mL.
NOTE:
Appropriate assay techniques include absorption spectrophotometry, radioimmunoassay, refractive index, and
conductivity.
Protein Retention and Concentrate Recovery, continued