Use of Ultrafiltration for Protein and Nucleic Acid Sample Preparation
Biomolecule purification involves a complex series of steps where molecules are selectively separated using size or a variety of other biochemical properties. Many steps require desalting and buffer exchange as well as sample concentration to prepare the biomolecule sample for the next step in purification. Desalting/buffer exchange can be a critical step in the process where yields and biological activity can be significantly reduced. It can be accomplished using methods such as gel filtration, size exclusion HPLC, dialysis, and ultrafiltration (UF). Compared to the use of non-membrane-based processes, filtration with UF has a number of technical advantages:

• Simultaneous Processing – desalting and sample concentration occur in the same step
• Speed and Simplicity – ultrafiltration is fast and easy to perform
• Consistency – the use of membranes eliminates problems with gel beds (e.g., irregular flow rates, channeling, variability in resin amount, dilution of sample)
• Biological Activity – no organic extraction is required and the ionic and pH milieu is maintained
• Low Cost – no special equipment required
• Versatility – useful at low and high-throughput modes, with or without automation, and at varying temperatures

To eliminate the problems experienced with traditional purification techniques, Pall Life Sciences has incorporated the same UF technology used in our centrifugal devices into our AcroPrep™ filter plates. Using UF to separate molecules based on size has proven to be one of the most effective ways to process biological samples. Although the primary basis for separation is molecular size, other factors such as molecule shape and charge also play a role. Molecules larger than the membrane pores are retained on the surface of the membrane, while smaller molecules pass through the membrane into the filtrate.

AcroPrep 96 UF Filter Plates
Pall Life Sciences has a long and successful history of making high performance ultrafiltration membranes and single use centrifugal devices containing these membranes. To address needs for automation, high throughput and the ability to use vacuum for filtration, Pall Life Sciences has developed the AcroPrep 96 filter plate containing Omega™ ultrafiltration membranes. Highlights of AcroPrep 96 UF filter plates include:

• Omega membrane is low in biomolecule binding and provides greater than 90% recoveries
• Polypropylene construction is chemically resistent and biologically inert providing solvent resistance and low biomolecule binding
• Rigid, single-piece construction
• Conforms to SBS guidelines for automation
• Individually sealed membranes prevent lateral flow and crosstalk
• Outlet tips and splash guards ensure clean filtrate collection
• Proprietary plate design minimizes solution/sample weeping
• Ability to simultaneously process up to 96 samples

Choosing the Correct MWCO
Ultrafiltration membrane pore sizes are expressed as Molecular Weight Cut Off (MWCO). Because different manufacturers use different molecules to define the MWCO of their membranes, it is important to perform pilot experiments to verify membrane performance in a particular application.

As a general rule, choose a UF membrane with a MWCO three to six times smaller than the molecular weight of the molecule to be retained. (See Tables 1 and 2 for detailed retention characteristics of various proteins and nucleic acids.) For most desalting purposes, we recommend using AcroPrep 96 with 10K membrane. A 10K MWCO will allow efficient removal of salts and detergents while maximizing recovery of most proteins and nucleic acid samples.

Table 1. Protein Recoveries For Different AcroPrep 96 MWCO Devices

	MW (kDa)	10K	30K	100K
Vitamin B12 - 200 µg/mL	1.3	4 + 1%	5 + 1%	7 + 2%
Ovalbumin, turkey - 1.0 mg/mL	45	97 + 2%	98 + 0%	48 + 7%
IgG, goat - 1.0 mg/mL	160	98 + 1%	98 + 0%	96 + 1%
Apoferritin, equine - 1.0 mg/mL	443	96 + 1%	89 + 1%	93 + 0%

300 µL of test protein solution were added to wells of AcroPrep 96 10, 30 and 100K plates. Each test plate was matched to a receiver plate and the assembly spun at 2,000 x g for 40 min. Following centrifugation, retentate samples were collected by adding 300 µL of buffer to each assay well, then allowing plate to stand at room temperature for 5 min. before pipetting up and down 10 times to remove sample to fresh tubes. Protein concentration was determined using UV spectrophotometric analysis. Two independent experiments were performed, n = 18. Error values represent standard error of the mean.

Table 2. Nucleic Acid Recoveries For Different AcroPrep 96 MWCO Devices

	Size (kDa)	10K	30K
20mer Oligonucleotide, 1 µg	6.5	2 + 8%	2 + 8%
41mer Oligonucleotide, 1 µg	13.3	15 + 36%	12 + 37%
137 bp pUC18 Fragment, 2 µg	91.1	81 + 9%	80 + 13%
214 bp pUC18 Fragment, 2 µg	142.3	79 + 8%	62 + 8%
537 bp pUC18 Fragment, 2µg	357.1	87 + 14%	87 + 8%

50 µL volumes of nucleic acid test solutions were added to AcroPrep 96 UF plates. Each test plate was matched with a receiver plate and vacuum filtered at 63.5 cm Hg (25 in. Hg). The retentate was recovered by adding 50 µL dH₂O to the assay wells and allowing solution to stand for 5 minutes at room temperature before pipetting up and down 10 times to remove sample to a fresh tube. Retentate, along with unprocessed controls, were mixed with 2X SybrGreenI to a final concentration of 1X in a solid bottom 96 well plate and analyzed with an automated fluorescence detector using a 485nm/520nm (excitation/emission) filter set. Error values represent standard deviation, n = 8.

Centrifugation

1. Optional conditioning, prewet membrane for maximal sample recovery.
   1. Place an AcroPrep 96 filter plate of the desired MWCO over a solid-bottom receiver plate¹.
   2. Add 20 – 100 µL of buffer/liquid (no solutes) into well of the AcroPrep 96 UF filter plate.
   3. Place filter and receiver plates together into a standard swinging bucket microtiter plate rotor assembly.
   4. Centrifuge at 2,000 x g for 5 minutes. (For approximate filtration times at other centrifugal speeds, see Table 3a.)
   5. After all fluid has evacuated from the well(s), discard receiver plate.
2. Place an AcroPrep 96 filter plate of the desired MWCO over a solid-bottom receiver plate¹.
3. Add biological sample(s), up to 350 µL per well.
4. Place filter and receiver plates together into a standard swinging bucket microtiter plate rotor assembly.
5. Centrifuge².
6. To recover protein or nucleic acid samples, add 20 – 300 µL of buffer to each well, pipet up and down several times, then collect liquid. (At this point, samples are generally > 90% desalted/buffer exchanged. See Table 4 for additional information.)
   Additional notes
   1. To achieve simple buffer exchange without a change in sample concentration, use the same or slightly less than the starting volume to recover samples.
   2. To achieve sample concentration, add a smaller volume than the starting volume when recovering the sample from the membrane.
7. Optional diafiltration: In order to further decrease the salt concentration and increase the purity of the sample, repeat steps 2-6. (Usually two cycles of buffer exchange will remove > 95% of salts, however, be warned that with each added diafiltration step, there is an increased risk of sample loss. Therefore, quality/purity versus yield considerations must be made.)

Vacuum Filtration

1. Optional conditioning, prewet membrane for maximal sample recovery.
   1. Place an AcroPrep 96 filter plate of the desired MWCO onto a vacuum manifold. (If the filtrate is to be saved, place a solid-bottom receiver plate into the vacuum manifold.)
   2. Add 20 – 100 µL of buffer/liquid (no solutes) into the wells of the AcroPrep 96 UF filter plate.
   3. Apply vacuum until all the liquid has evacuated from the wells³.
   4. After all fluid has evacuated from the well(s), discard receiver plate.
2. Place the AcroPrep 96 UF filter plate into vacuum manifold. (If the filtrate is to be saved, insert a new collection plate also into vacuum manifold.)
3. Add biological sample(s), up to 350 µL per well.
4. Apply vacuum³.
5. When most or all of the liquid has emptied, turn off and release vacuum. Do not release vacuum by pulling the corner of the plate as this can degrade the manifold gasket. If desired, tap plates gently to remove hanging drops from the bottom of the filter plate.
6. To recover protein or nucleic acid samples, add 20 – 300 µL of buffer to each well, pipet up and down several times, then collect liquid. (At this point, samples are generally > 90% desalted. See Table 4.)
   Additional notes
   1. To achieve simple buffer exchange without a change in sample concentration, use the same or slightly less than the starting volume to recover samples.
   2. To achieve sample concentration, add a smaller volume than the starting volume when recovering the sample from the membrane.
7. Optional diafiltration: In order to further decrease the salt concentration and increase the purity of the sample, repeat steps 2-6. (Usually two cycles of buffer exchange will remove > 95% of salts, however, be warned that with each added diafiltration step, there is an increased risk of sample loss. Therefore, quality/purity versus yield considerations must be made.)

Table 3. Sample Processing Times for 500 mM NaCl Using AcroPrep 96 10K

a. Centrifugation - Time to Evacuate Well (min:sec)

	500 x g	1,000 x g	2,000 x g	3,000 x g
20 µL	< 30:28 + 7:52	< 7:40 + 2:32	< 5:00 + 0	< 5:00 + 0
50 µL	< 31:34 + 5:36	< 9:32 + 1:29	< 5:00 + 0	< 5:00 + 0
100 µL	< 29:50 + 6:01	< 10:00 + 0	< 5:00 + 0	< 5:00 + 0
200 µL	< 30:10 + 5:00	< 12:11 + 2:31	< 5:00 + 0	< 5:00 + 0
300 µL	< 36:05 + 5:02	< 15:00 + 0	< 10:00 + 0	< 5:00 + 0

Indicated volumes of 500 mM NaCl solution were added to wells of each test plate in duplicate. Plates were matched with receiver plates and spun at 500 x g, 1,000 x g, 2,000 x g or 3,000 x g for increments of 5 minutes until complete evacuation was achieved. n = 16. Averages and standard deviations shown.

b. Vacuum - Time to Evacuate Well (min:sec)

	10 in. Hg	25 in. Hg
20 µL	< 1:52 + 0:23	< 0:51 + 0:08
50 µL	< 4:01 + 1:02	< 1:53 + 0:45
100 µL	< 8:23 + 2:09	< 4:24 + 1:07
200 µL	< 16:07 + 3:28	< 10:35 + 3:09
300 µL	< 22:37 + 3:49	< 15:03 + 2:17

Indicated volumes of 500 mM NaCl solution were added to wells of each test plate in duplicate. Plates were evacuated with vacuum at 25.4 cm Hg (10 in. Hg) or 63.5 cm Hg (25 in. Hg). n = 16. Averages and standard deviations shown.

Ultrafiltration is a reliable, gentle and trusted technique for the removal of salts or exchange of buffer components. The AcroPrep 96 filter plate containing UF membranes provides an excellent platform for high-throughput purification applications. With just a single filtration and resuspension cycle (no diafiltration), > 90% of the salt is removed (Table 4). With two diafiltration steps added, the purity increases to > 95% (Table 5).

Table 4. Desalting Efficiencies For Each Filtration Step (% Salt Removal)

Sample	Initial Filtration	1st Wash	2nd Wash	3rd Wash	Total
200 mM NaCl	97.0 + 0.2	2.0 + 0.1	0.2 + 0.03	Below Detection	99.2 + 0.1
500 mM NaCl	98.1 + 0.3	1.2 + 0.1	0.2 + 0.00	Below Detection	99.5 + 0.4
3 M (NH4)2SO4	97.9 + 0.7	0.6 + 0.04	0.1 + 0.02	Below Detection	98.6 + 0.7
50 mM EDTA	94.0 + 1.1	3.4 + 0.5	0.2 + 0.04	Below Detection	97.6 + 1.2
100 mM MgCl2	96.1 + 0.4	2.1 + 0.1	0.08 + 0.02	Below Detection	98.3 + 0.5
100 mM KOAc	92.2 + 0.3	1.8 + 0.04	0.05 + 0.01	Below Detection	94.0 + 0.3
500 mM KOAc	96.9 + 1.0	1.6 + 0.04	0.11 + 0.01	Below Detection	98.6 + 1.0

300 µL of test solution was added to wells of 10K plates. Test plates were matched with a receiver plate and spun at 2,000 x g for 40 min. Retained samples were washed by adding 300 µL dH₂O to each assay well followed by centrifugation for 25 minutes at 2,000 x g. The wash procedure was repeated for a total of three washes. Initial filtrate as well as filtrate from the three washes were collected and measured for salt concentration using a conductivity meter. The total salt removal was calculated by summing up the percentage of salt removal at each of the filtration steps. n = 5.

Table 5. Residual Salt Remaining After Two Buffer Exchanges

	Remaining [Salt]	Salt Removal
200 mM NaCl	2 mM	99.2 %
500 mM NaCl	2 mM	99.5%
100 mM KOAc	6 mM	94.0 %
500 mM KOAc	7 mM	98.6%

Remaining salt concentration in the wells was calculated by substracting the Total Percentage Salt Removal value (data from Table 4) from 100 to determine the percent salt remaining. The percent salt remaining was then converted to mM salt remaining by multiplying the percent salt remaining value with the starting concentration. n = 5.

In addition to desalting, ultrafiltration is also useful for the concentration of biomolecules. Throughout the process of using a UF membrane, the yields and recoveries remain high (Figure 1) allowing the researcher to have confidence that the sample will not only be effectively concentrated and desalted, but not lost in the process.

The efficiency of AcroPrep 96 UF filter plates to remove salts and other small molecules is not at the expense of sample recovery. Figure 1 demonstrates that using AcroPrep 96 10K, recovery of ovalbumin proteins (45 kDa) at two different concentrations, 0.1 and 1.0 mg/mL, were greater than 90% with salt removal efficiencies of also greater than 95%. Using BSA (66 kDa) and a 30K MWCO membrane, we observed similar protein recoveries and desalting effeciencies.

Figure 1. Desalting Ovalbumin and BSA Using AcroPrep 96 with 10 and 30K Membranes

300 µL of indicated protein solutions were added to wells of 10 or 30K plates. Each test plate was matched to a receiver plate and the assembly spun at 2,000 x g for 40 min. Following centrifugation, retained proteins were collected by adding 300 µL of buffer to each assay well, then allowing plate to stand at room temperature for 5 min. before pipetting up and down 10 times to remove sample to fresh tubes. Protein concentration was determined using UV spectrophotometric analysis (n = 3). The percentage of salt removal was determined using a conductivity meter per procedure described in Table 4. A representative experiment is shown here.

In summary, the AcroPrep 96 filter plates containing UF membranes provide a fast and simple means to efficiently desalt, buffer exchange and/or concentrate samples. The versatile multi-well format allows one to process up to 96 samples simultaneously, or as little as one sample at a time. AcroPrep 96 filter plates are compatible with most liquid handlers and vacuum manifolds⁴.

¹ Solid-bottom receiver plate compatible with AcroPrep 96 is Nunc™ 96 polypropylene microwell plate (conical well) PN 249944.

² Refer to Table 3a for approximate centrifugation times at various speeds.

³ For approximate filtration times at various vacuum pressures, see Table 3b.

⁴ AcroPrep 96 filter plates are compatible with a variety of currently available robotic systems and vacuum manifolds from the following manufacturers:

Liquid Handlers

• Adept Technology, Inc.
• Beckman Coulter, Inc.
• Cyber Lab
• Hamilton Company
• Matrix Technologies Corp.
• Oyster Bay Pump Works, Inc.
• PerkinElmer Inc.
• Porvair
• Qiagen Inc.
• Robbins Scientific Corp.
• Robocon
• Tecan U.S.
• Titantele
• Tomtec
• Velocity 11
• Waters

Vacuum Manifolds

• Pall Life Sciences
• 3M
• Innovative Microplates
• Millipore
• Qiagen
• Porvair
• Whatman

AcroPrep 96, 350 µL Volume Plates

Part Number	Product Description	Plate Color	Packaging
5034	AcroPrep 96, 10K	natural	10/pk
5035	AcroPrep 96, 30K	natural	10/pk
5036	AcroPrep 96, 100K	natural	10/pk

• Centrifugal Devices for nucleic acid and protein sample preparation in the following sample volumes:

Device	Sample Volume
Nanosep® Device	up to 0.50 mL
Microsep™ Device	up to 3.5 mL
Macrosep® Device	up to 15 mL
Jumbosep™ Device	up to 60 mL

• Nanosep MF (microfiltration) Centrifugal Devices are available with Bio-Inert or GHP membranes for low protein binding and high recoveries in applications such as particulate removal prior to sample analysis (GHP product is HPLC grade) and removal of precipitates.
• BioTrace™, Biodyne®, and Fluorotrans® Transfer Membranes offer precise performance and compatibility with nearly every detection system available.
• AcroWell™ and AcroPrep 96 and 384-well Filter Plates are an excellent platform for a wide variety of molecular biology, analytical, and high throughput sample preparation and detection applications.
• Vivid™ Gene Array Slides are microarray slides of unique membrane construction for high quality expression analysis.