Amicon ultra-0.5 Filter Devices User Manual

Amicon ultra-0.5 Filter Devices User Manual

Introduction

Millipore’s Amicon® Ultra-0.5 centrifugal filter devices provide fast ultrafiltration, 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 30-fold concentration. Typical processing time is 10 to 30 minutes depending on the Nominal Molecular Weight Limit
(NMWL). Solute polarization and subsequent fouling of the membrane are minimized by the vertical design, and a physical deadstop in the filter device prevents spinning to dryness and potential sample loss. Efficient recovery of the concentrated sample (retained species) is achieved by a convenient reverse spin step after collecting the filtrate. Amicon Ultra-0.5 devices are supplied non-sterile and are for single use only.

The Amicon Ultra-0.5 product line includes 5 different cutoffs (Nominal Molecular Weight Limit, NMWL). These devices are for research use only and not for use in diagnostic procedures.

• Amicon Ultra 3K device — 3,000 NMWL
• Amicon Ultra 10K device — 10,000 NMWL
• Amicon Ultra 30K device — 30,000 NMWL
• Amicon Ultra 50K device — 50,000 NMWL
• Amicon Ultra 100K device — 100,000 NMWL

Applications

• The concentration of biological samples containing antigens, antibodies, enzymes, nucleic acids (DNA/RNA samples, either single- or double-stranded), microorganisms, column eluates, and purified samples
• Purification 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, buffer exchange, or diafiltration

Materials Supplied

The Amicon Ultra-0.5 device is supplied with two microcentrifuge tubes. During operation, one tube is used to collect filtrate; the other to recover the concentrated sample.

Required Equipment

• Centrifuge with fixed angle rotor that can accommodate 1.5 mL microcentrifuge tubes CAUTION: To avoid damage to the device during centrifugation, check clearance before spinning.

Suitability
Preliminary recovery and retention studies are suggested to ensure suitability for the intended use. See the “How to Quantitate Recoveries” section.

Device Storage
Store at room temperature.

Specifications

• Maximum initial sample volume 500 μL

• Typical final concentrate volume 15–20 μL

• Recommended relative centrifugal force 14,000 × g for concentration spin 1,000 × g for reverse spin

• Maximum relative centrifugal force 15,000 × g

• Active membrane area 1 cm2

• Hold-up volume < 5 μL
  Dimensions
  

• Length (concentration mode; device in tube): 49.9 mm (1.96 in.)

• Length (reverse spin; device upside down in tube): 47.4 mm (1.87 in.)

• Tube (cap closed) Diameter: 10.8 mm (0.43 in.) Length: 42.1 mm (1.66 in.)

• Filter device Diameter: 9.4 mm (0.37 in.) Length: 29.5 mm (1.16 in.)
  Materials of Construction

• Filter devise Copolymer styrene/butadiene

• Membrane Millipore Ultracel® low binding regenerated cellulose

• Collection tube Polypropylene

The ultrafiltration membranes in Amicon Ultra-0.5 devices contain trace amounts of the following:

• Amicon Ultra 3K devices: glycerine
• Amicon Ultra 10K device: trimethylene glycol (TEG)
• Amicon Ultra 30K devices: glycerine
• Amicon Ultra 50K devices: glycerine
• Amicon Ultra 100K devices: glycerine

If these materials interfere with the analysis, pre-rinse the device with buffer or Milli-Q® water. If the 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 pre-rinsing, leave the fluid on the membrane until the device is used.

Chemical Compatibility

Amicon Ultra centrifugal devices are intended for use with biological fluids and aqueous solutions. Before use, check the sample for chemical compatibility with the device.

Table 1. Chemical Compatibility of Amicon Ultra Filter Devices

Acids

Alkalis

Detergents

• Contact with this chemical may cause materials to leach out of the component parts. Solvent blanks are recommended to determine whether leachables represent potential assay interferences.

Organic solvents

Miscellaneous

• Contact with this chemical may cause materials to leach out of the component parts. Solvent blanks are recommended to determine whether leachables represent potential assay interferences.

How to Use Amicon Ultra-0.5 Centrifugal Filter Devices

1. Insert the Amicon Ultra-0.5 device into one of the provided microcentrifuge tubes.
2. Add up to 500 µL of sample to the Amicon Ultra filter device and cap it.
3. Place capped filter device into the centrifuge rotor, aligning the cap strap toward the center of the rotor; counterbalance with a similar device.
4. Spin the device at 14,000 × g for approximately 10–30 minutes depending on the NMWL of the device used. Refer to Figure 1 and table 3 for typical spin times.
5. Remove the assembled device from the centrifuge and separate the Amicon Ultra filter device from the microcentrifuge tube.
6. To recover the concentrated solute, place the Amicon Ultra filter device upside down in a clean microcentrifuge tube. Place in a centrifuge, aligning the open cap towards the center of the rotor; counterbalance with a similar device. Spin for 2 minutes at 1,000 × g to transfer the concentrated sample from the device to the tube. The ultrafiltrate can be stored in the centrifuge tube.
   NOTE: For optimal recovery, perform the reverse spin immediately.

How to Quantitate Recoveries

Calculate total recovery, percent concentrate, and percent filtrate 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, refractive index, and conductivity.

Direct Weighing Procedure
The density of most dilute proteins is nearly equal to the density of water (i.e., 1 g/mL). Using this property, the concentrate and filtrate recoveries can be quantitated by weighing them and converting the units from grams to milliliters. This technique is valid only for solutions with concentrations of approximately 20 mg/mL or less.

1. Separately weigh the empty filter device, filtrate collection tube, and concentrate collection tube before use.
2. Fill filter device with a solution and reweigh.
3. Assemble the device in filtrate collection tube and centrifuge per instructions.
4. Collect the concentrate by a reverse spin into the pre-weighed concentrate tube.
5. Remove the device from the concentrate collection tube and weigh the filtrate and concentrate collection tubes.
6. Subtract weight of empty devices/tubes to calculate the weights of starting material, filtrate, and concentrate.
7. Assay the filtrate and concentrate to determine solute concentration.
8. Calculate recoveries using the weight/volume data and the measured concentrations as follows:
   % total recovery = % concentrate recovery + % filtrate recovery
   Wc = total weight of concentrate before assay Wo = weight of original starting material
   Wf = weight of filtrate
   Cc = concentrate concentration
   Co = original starting material concentration Cf = filtrate concentration

Performance – DNA Concentration

The Amicon Ultra-0.5 30K device provides the best balance between recovery and spin time for double-stranded DNA for base pairs ranging from 137 to 1159. To achieve maximum PCR product recovery and primer removal with primers greater than 20 bases, one or two additional spins with Tris-EDTA
(TE ) buffer are recommended.
Table 2. Typical recovery of nucleotides from Amicon Ultra-0.5 30K device

PCR Product (base pairs)| PCR Primer (bases)| PCR Recovery (%)| PCR Primer Removal (%)| TE Washes (number)
---|---|---|---|---
137| 10| ≥ 95| ≥ 90| 0
 | 20| ≥ 90| ≥ 85| 1
 | 48| ≥ 90| ≥ 75| 2
301| 10| ≥ 90| ≥ 90| 0
 | 20| ≥ 85| ≥ 90| 1
 | 48| ≥ 90| ≥ 80| 2
648| 10| ≥ 95| ≥ 90| 0
 | 20| ≥ 90| ≥ 90| 1
 | 48| ≥ 95| ≥ 90| 2
1159| 10| ≥ 90| ≥ 90| 0
 | 20| ≥ 90| ≥ 95| 1
 | 48| ≥ 95| ≥ 95| 2

Spin conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume, 20–30 μL final volume, 10-minute spin, n=12.

Flow Rate
Factors affecting flow rate include sample concentration, starting volume, chemical nature of solute, relative centrifugal force, centrifuge rotor angle, membrane type, and temperature. Figure 1 and Table 3 can be used to estimate the time required to achieve a given volume of filtrate or concentrate for a variety of protein markers. A typical spin time for a 500 µL sample in a fixed angle rotor is approximately 10–30 minutes (depending on device’s nominal molecular weight limit). While most of the sample is filtered in the first 5 to 10 minutes of centrifugation, the lowest concentrate volume (15–20 μL) is reached after spinning for 10–30 minutes.

Figure 1. Typical Filtrate Volume vs. Spin Time

Spin conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume. Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=8.

Table 3. Typical Concentrate Volume / Concentration Factor vs. Spin Time

Concentrate volume / Concentration Factor

3K device     10K device    30K device   50K device    100K devices

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| 215| 2| 66| 7| 42| 12| 28| 18| 58| 9
10| 114| 4| 35| 14| 23| 22| 20| 25| 19| 26
15| 80| 6| 22| 22| 19| 27| 17| 30| 15| 33
20| 62| 8| 20| 24| 17| 30| 15| 33| 13| 36
30| 48| 10| 15| 31| 15| 32| 15| 36| 11| 41

Spin conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 μL starting volume. Protein markers used: Cytochrome c for 3K and 10K, BSA for 30K and 50K, and IgG for 100K, n=12. Shaded volumes were used for the calculation of protein recovery in Table 4.

Protein Retention and Concentrate Recovery
The membranes used in Amicon Ultra devices are characterized by a nominal molecular weight limit (NMWL); that is, their ability to retain molecules above a specified molecular weight. Solutes with molecular weights close to the NMWL 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. Millipore recommends using a membrane with an NMWL at least two times smaller than the molecular weight of the protein solute that one intends to concentrate. Refer to Table 4 for “Typical Retention of Protein Markers”.

Table 4: Typical Retention of Protein Markers

Marker/Concentration| Weight| NMWL| % Retention| (min)
---|---|---|---|---
α-Chymotrypsinogen (1 mg/mL)| 25,000| 3K| > 95| 30
Cytochrome c (0.25 mg/mL)| 12,400|  | > 95| 30
Vitamin B-12 (0.2 mg/mL)| 1,350|  | < 42| 30
α-Chymotrypsinogen (1 mg/mL)| 25,000| 10K| > 95| 15
Cytochrome c (0.25 mg/mL)| 12,400|  | > 95| 15
Vitamin B-12 (0.2 mg/mL)| 1,350|  | < 42| 15
BSA (1 mg/mL)| 67,000| 30K| > 95| 10
Ovalbumin (1 mg/mL)| 45,000|  | > 95| 10
Cytochrome c (0.25 mg/mL)| 12,400|  | < 35| 10
BSA (1 mg/mL)| 67,000| 50K| > 95| 10
Ovalbumin (1 mg/mL)| 45,000|  | ~ 40| 10
Cytochrome c (0.25 mg/mL)| 12,400|  | < 20| 10
Thyroglobulin (0.5 mg/mL)| 677,000| 100K| > 95| 10
IgG (1 mg/mL)| 156,000|  | > 95| 10
Ovalbumin (1 mg/mL)| 45,000|  | < 30| 10

Factors that determine sample recovery include the nature of the protein solute relative to the device NMWL chose, starting concentration, and concentration factor. Table 5 provides typical recoveries for Amicon Ultra-0.5 devices.
Table 5. Typical Concentrate Recovery

Marker/
Concentration

Cytochrome c

(0.25 mg/mL)

| Molecular
Weight

12,400

| Device
NMWL

3K

| Spin Time
(min )

30

| Concentrate
Volume (μL)

48

| Concentration
Factor (x)

10

| Concentrate
Recovery (%)

98

---|---|---|---|---|---|---
Cytochrome c

(0.25 mg/mL)

| 12,400| 10K| 15| 22| 22| 93
BSA (1 mg/mL)| 67,000| 30K| 10| 23| 22| 97
BSA (1 mg/mL)| 67,000| 50K| 10| 20| 25| 92
IgG (1 mg/mL)| 156,000| 100K| 10| 19| 26| 92

Spin Conditions: 40° fixed angle rotor, 14,000 × g, room temperature, 500 µL starting volume, n=12. The shaded volumes were taken from Table 3.

Maximizing Sample Recovery

Low sample recovery in the concentrate may be due to adsorptive losses, over- concentration, or passage of the 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 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 NMWL Amicon Ultra-0.5 device.

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-0.5 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 micro solute has been sufficiently reduced. See the example below.

Centrifugal Product Ordering Information

Initial volume (mL)| Final concentrate volume (µL)|

Product

| ****

Qty /

pk

| ****

3,000

NMWL

| ****

10,000

NMWL

| ****

30,000

NMWL

| ****

50,000

NMWL

| ****

100,000

NMWL

---|---|---|---|---|---|---|---|---
 |  | Amicon| 8| UFC500308| UFC501008| UFC503008| UFC505008| UFC510008
0.5| 20| Ultra-0.5

device

| 24

96

| UFC500324

UFC500396

| UFC501024

UFC501096

| UFC503024

UFC503096

| UFC505024

UFC505096

| UFC510024

UFC510096

 |  |  | 500| UFC5003BK| UFC5010BK| UFC5030BK| UFC5050BK| UFC5100BK
Amicon Ultra-0.5 Collection Tubes| 96| UFC50VL96|
 |  | Amicon| 8| UFC800308| UFC801008| UFC803008| UFC805008| UFC810008
4| 30–70| Ultra-4| 24| UFC800324| UFC801024| UFC803024| UFC805024| UFC810024
 |  | device| 96| UFC800396| UFC801096| UFC803096| UFC805096| UFC810096
 |  | Amicon| 8| UFC900308| UFC901008| UFC903008| UFC905008| UFC910008
15| 150–300| Ultra-15| 24| UFC900324| UFC901024| UFC903024| UFC905024| UFC910024
 |  | device| 96| UFC900396| UFC901096| UFC903096| UFC905096| UFC910096

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70

|

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|

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|

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|

N/A

|

UFC710008

---|---|---|---|---|---|---|---|---

0.5

|

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| MultiScreen® 96 well plate/ Ultracel-10 membrane|

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Technical Assistance

For more information, contact the Millipore office nearest you. In the U.S., call
1-800-MILLIPORE (1-800-645-5476). Outside the U.S., see your Millipore catalog for the phone number of the office nearest you or go to our website at www.millipore.com/offices for up-to- date worldwide contact information. You can also visit the tech service page on our web site at www.millipore.com/techservice.

Standard Warranty

Millipore Corporation (“Millipore”) warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products. MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications, and descriptions of Millipore products appearing in Millipore’s published catalogs and product literature may not be altered except by an express written agreement signed by an officer of Millipore. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and if given, should not be relied upon.

In the event of a breach of the foregoing warranty, Millipore’s sole obligation shall be to repair or replace, at its option, the applicable product or part thereof, provided the customer notifies Millipore promptly of any such breach. If after exercising reasonable efforts, Millipore is unable to repair or replace the product or part, then Millipore shall refund to the customer all monies paid for such applicable product or part. MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL, OR ANY OTHER INDIRECT DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY CUSTOMER FROM THE USE OF ITS PRODUCTS.

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