Protein Sample Preparation and Analysis Application Manual - Pre-Analytical Applications
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Protein Fractionation (Complexity Reduction)
- Protein Fractionation (Complexity Reduction) Using Q, S, DEAE, and CM Ion Exchange Media (2.2.2)
- Affinity Separations – Using Chromatography Resins for Protein Fractionation (Complexity Reduction) (2.2.3)
- Protein Fractionation (Complexity Reduction) Using Mixed Mode Chromatography Resins (2.2.4)
- Protein Fractionation (Complexity Reduction) Using Size Fractionation Chromatography Resins (2.2.5)
Introduction
Sample complexity reduction is an important first step to facilitating access to the low abundant proteins of interest for clinical research and diagnostics. The process for human serum and plasma frequently includes the depletion of highly abundant proteins such as albumin and IgG in combination with other fractionation technologies, such as ion exchange or affinity separation, prior to 2D gel or LC-MS/MS analyses.
Pall currently offers several solutions for small-scale chromatographic protein fractionation (see table below). Options include chromatography resin for affinity capture, ion exchange separation, and mixed-mode capture resins. Pall also offers membrane chromatography solutions, i.e., Mustang membranes. All chemistries are available for use in several forms including standard chromatography columns, disposable columns, spin filters, and multi-well filter plates.
Protein Fractionation Quick Selection Guide
Available Separation Columns
| Description | Column Volume | Available from Pall |
|---|---|---|
| Glass Chromatography Column | Varies | No |
| Disposable Chromatography Column | Varies | No |
| Spin Filter | < 1 mL | Yes |
| Deep Well Multi-Well Filter Plate | 96 x < 1 mL | Yes |
| Multi-Well Filter Plate | 96 x < 350 µL | Yes |
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Concentration, Desalting, and Buffer Exchange
- Concentration and Desalting Using Ultrafiltration (2.4.2)
- Desalting with Size Exclusion Media (2.4.3)
Introduction
Biomolecule purification involves a complex series of steps where targets are selectively separated through sequential processes. The processes by which separation is performed often creates a need for the sample to be desalted or concentrated to prepare the biomolecule sample for the next step in the purification process.
Pall offers several technologies to perform efficient sample concentration, desalting, and buffer exchange including ultrafiltration (UF) spin filters, UF multi-well filtration plates, and bottled gel filtration media. Refer to the table below for a selection of UF products available.
UF Products for Concentration and Desalting
| Device | Sample Volume |
|---|---|
| AcroPrep 384 filter plate | < 100 µL |
| AcroPrep 96 filter plate | < 350 µL |
| Nanosep® device | < 0.5 mL |
| Microsep™ device | 0.5-3.5 mL |
| Macrosep® device | 3-15 mL |
| Jumbosep™ device | 15-60 mL |
Compared to other methods, UF methods for concentration and desalting offer a number of advantages including:
- Reduction of processing steps – desalting and concentration take place in a single step.
- Consistency – the use of UF products eliminates problems with gel beds.
- Versatility – UF is useful in both low and high throughput modes.
Pall also offers chromatography resins for desalting applications including Ultrogel AcA 202 and Trisacryl GF-05 resins. Refer to the below table for general properties.
Properties of Ultrogel AcA and Trisacryl GF-05 Resin
| Specification | Ultrogel AcA 202 Resin | Trisacryl GF-05M Resin |
|---|---|---|
| Particle Size | 60-140 µm | 40-80 µm |
| Monomer | 20% (w/v) Acrylamide | N-acryloyl-2-amino-2-hydroxymethyl-1,3-propanediol |
| Cross-linker | 2% (w/v) Agarose | Hydroxylated acrylic bifunctional monomer |
| Exclusion Limit | 22,000 dalton | 3,000 dalton |
| Linear Fractionation Range | 1,000-15,000 dalton | 200-2,500 dalton |
| Resolving Power (plates/m) | 3,000 | 2,500 |
| Working pH Range | 3-10 | 1-11 |
Pall Centrifugal Spin Filters
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Generic Clarification of Samples by Microporous Filtration (Particulate Removal)
- Clarification of 0.1-1.0 mL Samples Using AcroPrep 96 and 384 Multi-Well Filter Plates (2.5.2)
- Clarification of Samples (< 1 mL) in Spin Filters (Nanosep® Centrifugal Devices) (2.5.3)
- Clarification of Samples (1-100 mL) in an Acrodisc® Syringe Filter (2.5.4)
- Filtration of Reagents (> 50 mL) with a VacuCap® Vacuum Filtration Device (2.5.5)
- Clarification of Plasma or Serum Samples to Remove Cryoprecipitate Using Acrodisc Syringe Filters with a Prefilter (2.5.6)
- Prefiltration of Plasma or Serum Samples Using Acrodisc Syringe Filters with Glass Fiber (2.5.7)
Introduction
Although a basic filtration concept, the clarification and prefiltration of samples remains an important function within protein sample preparation applications. When filtration is used as a prefilter, matching the proper filter media and device to the application is critical. Large pore size filter materials are used to filter solutions prior to more detailed analysis. When selecting the best product for your application, several factors need to be considered. Sample viscosity, sample volume, and sample recovery are just some of the aspects that will drive the selection of the optimal device.
Pall offers a number of media and device options for fast, effective filtration with minimal sample hold-up for both single and high throughput sample processing. From sample volumes of a few microliters to multiple liters, Pall can supply the best product solution for your application.
Choose the Best Clarification Device for Your Needs
Acrodisc Syringe Filters for Analytical Sample Preparation
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