Clarification of 0.1-1.0 mL Samples Using AcroPrep™ 96 and 384 Multi-Well Filter Plates 2.5.2

Small-volume multiple samples (0.1–1.0 mL) can be clarified in parallel by microfiltration in the AcroPrep 96 and 384 multi-well filter plates. These filter plates, made to SBS standards, are available in 0.2, 0.45, and 1.2 μm pore sizes and in depth filter/microporous membrane combinations employing 1.0 and 3.0 μm pore size glass fiber media or a 54 μm screen material. The membranes available are:

• GHP, a modified hydrophilic polypropylene membrane
• Hydrophilic, low protein binding Supor® PES, polyethersulfone membrane
• Bio-Inert®, a modified low protein binding nylon membrane
• PTFE hydrophobic membrane 
• Glass fiber and glass fiber prefilter combinations

For viscous or heavy particle-containing samples, combination filters are available with integrated prefiltration membranes such as glass fiber prefilter over Bio-Inert membrane and polypropylene (PP) over Supor PES. The AcroPrep 96 and 384 multi-well filter plate properties are summarized in Table 2.56.

Table 2.56

Properties of the AcroPrep™ 96 and 384 MF Multi-Well Filter Plates

	Parameter
Specification	96-well 350 μL  Format	96-well 1 mL  Format	384-well 100 μL  Format
Materials of   Construction      Membrane    Device	Bio-Inert® (modified low protein-binding Nylon) GHP (hydrophilic    polypropylene) PTFE (hydrophobic PTFE) Glass fiber (borosilicate    glass without binder) Supor Polypropylene	GHP (hydrophilic polypropylene) PTFE (hydrophobic PTFE) Glass fiber (borosilicate glass without binder) Supor®  Polypropylene
Effective Membrane Area	0.25 cm2	0.05 cm2
Dimensions      Length      Width      Height (with Lid)      Height (without Lid)      Tip Length	12.78 cm (5.03 in.) 8.51 cm (3.35 in.) 1.66 cm (0.655 in.) 1.44 cm (0.565 in.) 0.53 cm (0.21 in.)	12.78 cm (5.03 in.) 8.51 cm (3.35 in.) 3.35 cm (1.32 in. 2.87 cm (1.13 in.) 0.53 cm (0.21 in.)	12.78 cm (5.03 in.) 8.51 cm (3.35 in.) 1.66 cm (0.655 in.) 1.44 cm (0.565 in.) 0.41 cm (0.16 in.)
Capacities      Maximum Well         Volume      Recommended        Volume      Hold-up Volume        (Membrane/Support)	0.35 mL 0.30 mL < 0.014 mL	1.0 mL 0.9 mL < 0.018 mL	0.1 mL 0.08 mL < 0.004 mL
Maximum Centrifugal       Force	3,000 x g
Centrifuge	Swinging bucket rotors
Operating Vacuum	25.4 cm Hg (10 in. Hg)	50.8 cm Hg (20 in. Hg)

Multi-well filter plates have a wide variety of applications and are key to some analytical and screening processes in biosciences. An application guideline for selection of membrane types is provided in Figure 2.51 and a listing of some applications arranged by membrane type is summarized in Table 2.57.

Figure 2.51

Filter Plate Quick Selection Guide

Table 2.57

Application Summary for AcroPrep™ Multi-Well MF Filtration Plates – Organized by Membrane Type

GHP Membrane	Cleavage reactions for combinatorial chemistry library screens Receptor-ligand screens Cell- and bead-based receptor-ligand screens HPLC sample prep and clarification Metabolic studies Micro-volume parallel filtration Molecular synthesis reactions Enzyme assays In-gel digestion Fluorescence assays Protein scouting – mini columns Processing of bead bound targets
Supor® Membrane	Lysate clearance Protein scouting – mini columns Processing of bead bound targets General filtration
PTFE Membrane	Molecular synthesis reactions using harsh reagents Drug synthesis reactions Solubility studies Cleavage reactions for combinatorial chemistry library screens Bead-based applications
Glass Fiber Media	Prefiltration of high solid and viscous samples Lysate clarification Radio labeled cell assays Enzyme assays
Glass Fiber over Bio-Inert®  Membrane	Clarification of cell lysate and tissue homogenates Preparation of proteinaceous solutions that may have a high solid content Applications that require prefiltration due to high sample solid or viscosity
Polypropylene over Supor   Membrane	Fast lysate clearance at low salt conditions Clarification of high particulate-laden solutions Preparation of proteinaceous solutions that may have a high solid content
54 μm Polyester Screen	Sample preparation for cell analysis/flow cytometry assays Separation of cell, organisms and large particulates

Protocol for AcroPrep™ 96 and 384 Multi-Well Filter Plate for 0.1-1.0 mL Samples

A. Materials Required

1. AcroPrep 96 and 384 filter plate with MF membranes. For specifications, see Table 2.56.
2. Collection plates [(Axygen 96-well polypropylene V-bottom, 0.5 mL (PN P96450V); or round bottom, 1.64 mL (PN PDW20); or 384-well polypropylene, 0.24 mL (PN P384240SQ)]
3. High purity water or buffer, such as phosphate buffered saline (PBS)
4. Separation apparatus
   1. Source of vacuum [25.4–50.8 cm Hg (10–20 in. Hg) (Pall vacuum manifold PN 5017)]
   2. Centrifuge fitted with a swinging bucket rotor with multi-well plate adapters and ability to spin up to 3,000 x g

B. Vacuum Manifold Filtration

1. During use, hold the plate so that the outlets on the bottom of the plate are not touched to prevent liquid flow due to wicking. 
2. Place a receiver plate into the vacuum manifold sized to collect the volume of the filter plate.
3. Place the filter plate on the vacuum manifold. Add 0.1 mL water or buffer.
4. Apply vacuum to manifold to initiate liquid flow. Recommended vacuum is 25.4 cm Hg (10 in. Hg) for 96-well plates.
   Tip: Do not exceed 38.1 cm Hg (10 in. Hg). Most house vacuum and aspirators do not exceed 38.1 cm Hg (15 in. Hg). For 384-well plates, vacuum up to 50.8 cm Hg (20 in. Hg) can be used.
5. Release vacuum and discard the first filtrate.
   Tip: Do not release vacuum by pulling the corner of plate. The maifold gasket will ddegrade.
6. Add the sample to be filtered to the plate. Incubate if required.
7. Gently tap the plate to remove any hanging droplets.
8. Release vacuum from the manifold and recover filtrate from the collection plate.
9. If needed, additional water or buffer solutions can be used. Collect wash filtrate separately and pool with first filtrate as needed.
   Tip: Some sample dilution may occur and the resulting filtrate may require reconcentration using an AcroPrep 96 or 384 filter plate with a 10K MWCO UF membrane.

C. Filtration Facilitated by Centrifugation in a Swinging Bucket Rotor

1. Place the AcroPrep 96 or 384 filter plate on top of a receiver plate sized to collect the volume of the filter plate.
2. Insert the two plates into a standard multi-well filter plate swinging bucket rotor assembly.
3. Place a duplicate pair of plates matching the weight of the test plate (add water to the receiver plate and match weight of the test plate).
   Tip: An imbalance can result with a single test plate if no counterbalancing plate is used. If different volumes of sample are used in multiple plates, they will need to be balanced in pairs by addition of water to empty wells.
4. Centrifuge at 500-3,000 x g for 1-2 minutes.
   Tip: The centrifugal force and time parameters can be varied to optimize the filtration rate of fluids in contact in the well of the plate.
5. If needed, additional water or buffer solutions can be used. Collect wash filtrate separately and pool with first filtrate as needed. Tip: Some sample dilution may occur and the resulting filtrate may require reconcentration using an AcroPrep™ 96 or 384 filter plate with a 10K MWCO UF membrane.

Application Data for AcroPrep 96 and 384 Multi-Well Filter Plate for 0.1-1.0 mL Samples

The modified polypropylene GHP membrane exhibits very low DNA and protein non-specific binding. An example of a comparison of macromolecule binding to GHP and a high binding nylon Biodyne® B membrane are summarized in Figure 2.52. The data clearly shows very low DNA or protein binding to the hydrophilic modified polypropylene membrane. 

Figure 2.52

Hydrophilic Modified Polypropylene (GHP) Membrane Is Extremely Low in DNA and Protein Non-Specific Binding

125I-labeled BSA (1.6 μg) or 32P-labeled DNA (500 ng) were diluted to 5 mL in PBS (BSA) or Tris-EDTA buffer, pH 8.0 (DNA), and filtered through a 13 mm disc of the indicated membrane. Filtration was carried out using a 10 mL syringe at a flow rate of 1.0 mL/min. Binding was determined by comparing the amount of radioactivity remaining in the membrane (triplicate) to the activity of the starting material by placing the disc or solution directly into a scintillation cocktail and counting in a scintillation counter. Biodyne B membrane is designed for biomolecules binding and was used as a positive control.

The construction of the multi-well filter plate leads to reduced “crosstalk” between adjacent wells during processing of parallel samples. An example of an experiment designed to measure “crosstalk” is summarized in Figure 2.53. The results clearly show no observable “crosstalk” between wells on the plate.

Figure 2.53

Observed Crosstalk in AcroPrep™ 96 Filter Plate

Fluorescein dye (0.200 mL of a 2 μg/mL in water stock) was added to wells of AcroPrep 96 filter plates with GHP membrane (5 plates total) in a checkerboard pattern. Alternate wells were filled with 0.200 mL of water. The fluid was evacuated from each filter plate using vacuum filtration at 30 cm Hg (12 in. Hg) for 15 seconds and the filter plate was read in a PerkinElmer, Wallac VICTOR^♦1420 Multilabel Counter. Wells filled with water that show a CPS reading above the dashed line (5.2 x 104 CPS, which is = “Average” Background + 5% Signal) in the graph constitute a crosstalk event. CPS = Counts Per Second.

An example of the impact of an AcroPrep filter plate on a Thyroid Stimulating Hormone (TSH) immunoassay application was presented at the Planet xMAP Symposium in April 2005 in conjunction with Luminex Corp. This study showed that the Pall Life Sciences AcroPrep 96 filter plates with 1.2 μm Supor® membrane show a significant reduction in the presence of false positives compared to competitive filter plates when used with the Luminex xMAP technoloy for serological TSH immunoassays. An example of this reduced occurrence of false positives is reproduced in Figure 2.54. In all lots of microspheres tested, the AcroPrep filter plates exhibited a marked reduction in non-specific reactivity than competitive plates.

Figure 2.54

Reduced Occurrence of False Positives in Serological TSH Immunoassays with AcroPrep 96 Filter Plates

Utilizing multiple lots of xMAP microspheres (Luminex Corp) in both AcroPrep 96 filter plates (green bars) and Competitor M filter plates (orange bars), serological immunoassay results were read using a Luminex LX100 instrument. The responses represent the reactivity towards the microspheres without proteins coupled to them which in turn gives an indication of false positives or ‘non-specific’ reactivity by the microspheres. For all lots of microspheres tested, the AcroPrep 96 filter plates exhibited a reduction in non-specific reactivity when compared to the competitive filter plate.

METHOD: Pipette 10 μL of microspheres to each well of the filter plate. Add 20 μL of 1:20 diluted serum sample (in PBS-4% BSA). Incubate for 1 hour at room temperature. Apply vacuum to the filter plate and wash each well twice with 100 μL PBS-1% BSA. After washing, apply 50 μL of target conjugated antibody to IgG to each well and incubate for 1 hour. Using vacuum, wash each well twice with 100 μL of buffer per wash. After washing, add 100 μL of buffer to each well, mix gently, and insert the plate into the LX100 instrument for reading.

Ordering Information for AcroPrep™ 96 and 384 Multi-Well Filter Plate for 0.1-1.0 mL Samples

AcroPrep 96 Filter Plates, 350 μL Well – MF Filter < 1.0 μm Pore Size

Part Number	Description	Pkg
5045	0.2 μm GHP membrane, natural	10/pkg
5030	0.45 μm GHP membrane, natural	10/pkg
5043	0.45 μm GHP membrane, white	10/pkg
5044	0.45 μm GHP membrane, black	10/pkg
5042	0.2 μm Bio-Inert® membrane, natural	10/pkg
5037	0.2 μm PTFE membrane, natural	10/pkg
5038	0.45 μm PTFE membrane, natural	10/pkg
5029	0.45 μm Supor® membrane, natural	10/pkg

AcroPrep 96 Filter Plates, 350 μL Well – MF and Screen Filters > 1.0 μm Pore Size

Part Number	Description	Pkg
5031	1.0 μm glass fiber, natural	10/pkg
5032	1.0 μm glass fiber, white	10/pkg
5039	1.2 μm Supor membrane, natural	10/pkg
5041	Prefilter material 1.2 μm Supor membrane, natural	10/pkg
5046	3.0 μm glass fiber media, 0.2 μm Bio-Inert membrane, natural	10/pkg
7267	30-40 μm PP/PE mesh screen, natural	10/pkg

AcroPrep™ 96 Filter Plates, 1 mL Well – MF Filter < 1.0 μm Pore Size

Part Number	Description	Pkg
5052	0.2 μm GHP membrane, natural	5/pkg
5054	0.45 μm GHP membrane, natural	5/pkg
5055	0.2 μm PTFE membrane, natural	5/pkg
5056	0.45 μm PTFE membrane, natural	5/pkg

AcroPrep 96 Filter Plates, 1 mL Well – MF and Screen Filters > 1.0 μm Pore Size

Part Number	Description	Pkg
5051	1.0 μm glass fiber media, natural	5/pkg
5053	3.0 μm glass fiber media/0.2 μm Bio-Inert® membrane, natural	5/pkg

AcroPrep 384 Filter Plates, 100 μL Well – MF Filter < 1.0 μm Pore Size

Part Number	Description	Pkg
5070	0.45 μm GHP membrane, long tips, natural	10/pkg
5071	0.45 μm GHP membrane, short tips, natural	10/pkg
5084	1.2 μm Supor® membrane, long tips, natural	10/pkg
5085	1.2 μm Supor membrane, short tips, natural	10/pkg

AcroPrep 384 Filter Plates, 100 μL Well – MF and Screen Filters > 1.0 μm Pore Size

Part Number	Description	Pkg
5072	1.0 μm glass fiber media, long tips, natural	10/pkg
5072W	1.0 μm glass fiber media, long tips, white	10/pkg
5073	1.0 μm glass fiber media, short tips, natural	10/pkg
5073W	1.0 μm glass fiber media, short tips, white	10/pkg
5084	1.2 μm Supor membrane, long tips, natural	10/pkg
5085	1.2 μm Supor membrane, short tips, natural	10/pkg

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