Gelfree 8100 Fractionation System

Description
Source

Gelfree^® 8100 Fractionation System

Sample complexity necessitates extensive fractionation for in-depth proteomic analysis using mass spectrometry. Protein Discovery, now part of the Expedeon group, has developed a commercial system using a novel technology, termed Gel Elution Liquid Fraction Entrapment Electrophoresis (GELFREE), for molecular weight-based fractionation of intact proteins with liquid phase recovery. Eight samples are run in parallel using a programmable control module in ca. 90 minutes. The system is shown to provide robust fractionation over the mass range 3.5 kDa - 500 kDa with high loading capacity, reproducibility, and recovery.

The Gelfree 8100 Fractionation System for mass spectrometry sample preparation partitions complex protein mixtures into userselectable liquid-phase molecular weight fractions.

Features

Intact protein molecular weight fractionation, isolation, and purification
Liquid-phase recovery without band or spot cutting
Broad mass range fractionation up to 500 kDa
Up to eight samples processed in parallel
Programmable fractionation for isolating and purifying targeted proteins
High protein recovery (>80%)
High reproducibility (<15% CV)
Sampling unbiased by hydrophobicity, pI
High loading capacity (>5X more than a 1D gel)
Proteins are recovered intact, for complete characterization

Applications

Simplify and reduce the dynamic range of complex protein mixtures for bottom-up discovery proteomics using LC-MS/MS
Fractionate and recover proteins intact for top-down proteomics
Isolate and enrich user-selected molecular weight fractions for targeted protein quantification using LC-MS/MS
Isolate intact proteins to analyze variants, post-translational modifications, alterations
Separate protein pull-down components for target protein purification
Separation, isolation, and intact recovery of antibodies intact for in-depth characterization

Components

The Gelfree 8100 Fractionation System consists of the Gelfree 8100 Fractionation Station and ready-to-use Gelfree 8100 Cartridges.

The Fractionation Station

The compact, bench-top Gelfree 8100 Fractionation Station provides specified voltage to each of eight independent channels and pauses for fraction collection according to user programming. Using the touch screen interface, the fractionation sequence is defined or selected, permitting broad mass range analyte fractionation or target protein enrichment in accordance with the users requirements. During operation, the touch screen interface provides feedback on the status and current/voltage that is applied for each channel.

The Cartridge Kits

Ready-to-use Gelfree 8100 Cartridge Kits complement the Gelfree 8100 Fractionation Station. Each Cartridge Kit contains pre-formulated Running and Sample Buffers, as well as the Fractionation Cartridge. The Gelfree 8100 Fractionation Cartridge contains eight independent channels for molecular weight fractionation and liquid phase recovery. All eight channels may be used simultaneously, or one or more channels may be used, saving the unused channels for use at a later time.

Four separate Gelfree 8100 Cartridge Kits are available for protein isolation, purification and fractionation across distinct mass ranges.

TheGelfree 8100 12% Tris-Acetate Cartridge Kitis designed for separation in the mass range 3.5 50 kDa, with resolution between 10 kDa and 50 kDa. The following image shows an aliquot of each of 12 Gelfree fractions from a yeast lysate partitioned using the 12% Cartridge Kit and run out on a 1D gel for visualization.

Fractionation of 500 μg Yeast Lysate using the Gelfree 8100 12% Tris-Acetate Cartridge Kit

TheGelfree 8100 10% Tris-Acetate Cartridge Kitis designed for separation in the mass range 3.5 100 kDa, with resolution between 15 kDa and 100 kDa.The following image shows an aliquot of each of 12 Gelfree fractions from a yeast lysate partitioned using the 10% Cartridge Kit and run out on a 1D gel for visualization.

Fractionation of 200 g Yeast Lysate using the Gelfree 8100 10% Tris-Acetate Cartridge Kit

Fractionation of 200 μg Yeast Lysate using the Gelfree 8100 10% Tris-Acetate Cartridge Kit

TheGelfree 8100 8% Tris-Acetate Cartridge Kitis designed for separation in the mass range 3.5 150 kDa, with resolution between 35 kDa and 150 kDa. The following image shows an aliquot of each of 12 Gelfree fractions from a yeast lysate partitioned using the Gelfree 8100 8% Tris-Acetate Cartridge Kit and run out on a 1D gel for visualization.

Fractionation of 500 μg Yeast Lysate using the Gelfree 8100 8% Tris-Acetate Cartridge Kit

The Gelfree 8100 5% Tris-Acetate Cartridge Kitis designed for separation in the mass rangeis designed for separation in the mass range 3.5 500 kDa, with resolution between 75 kDa and 500 kDa. The following image shows an aliquot of each of 12 Gelfree fractions from a yeast lysate partitioned using the Gelfree 8100 5% Tris-Acetate Cartridge Kit and run out on a 1D gel for visualization.

Fractionation of 200 μg Yeast Lysate using the Gelfree 8100 5% Tris-Acetate Cartridge Kit

How it Works

Gelfree technology is "Gel-eluted Liquid Fraction Entrapment Electrophoresis.Each of the eight Gelfree 8100 Cartridge channels consists of a precision-cast gel column surrounded by pipette-accessible sample loading and fraction collection chambers. Samples are mixed with the pre-formulated Sample Buffer and pipetted into the sample loading chamber. When electric current is applied, charged molecules in the channel migrate first focus in the stacking gel and then separate according to their electrophoretic mobility or, when using SDS, molecular weight. Smaller proteins move more quickly and elute into the fractionation collection chamber, where they are trapped in a defined liquid volume. At predefined intervals, the instrument will then automatically pause for easy removal of the liquid fraction with a pipette. For collection of the next size-based fraction, the sequence is restarted and the process continued.

Performance

The Gelfree 8100 system permits effective, programmable fractionation, isolation, and purification across a broad mass range. Combined with its high load capacity and high, liquid-phase recovery , the system provides the highest sensitivity of any pre-fractionation technology. The Gelfree may be operated in fractionation mode, in which the entire sample is partitioned into 1-29 selectable MW fractions, or in targeted isolation/purification mode, allowing for extraction of analytes in one or more pre-determined mass ranges.

Broad Mass Range Fractionation

The Gelfree 8100 Fractionation System offers molecular weight-based fraction targeting over a broad mass range. Using the 12% Cartridge Kit, the system is capable of partitioning complex protein samples across the mass range 10 50 kDa. Using the 10% Cartridge Kit, the system is capable of partitioning complex protein samples across the mass range 15 100 kDa. Using the 8% Cartridge Kit, the system is capable of partitioning complex protein samples across the mass range 35 150 kDa. Using the 5% Cartridge Kit, the system is capable of partitioning complex protein samples across the mass range 75 500 kDa. Recovery across all mass ranges is > 80%.

Intact protein molecular weight-based fractionation with liquid phase recovery

Recovery and Reproducibility

The Gelfree 8100 Fractionation System uses specialized materials to optimize protein recovery and prevent non-specific loss. In addition, each gel column used in the Gelfree system is comprised of a proprietary composite gel matrix that is robotically cast using state-of-the-art automation. Every gel tube is computer imaged to ensure compliance with strict quality control specifications and each cartridge is barcoded for tracking. This level of quality control and precision-manufacturing uniquely permits the absolute elution time reproducibility provided by the Gelfree system.

Gelfree Reproducibility of Fractionation. 200 μg yeast lysate prepared using the Gelfree 8100 Fractionation System. Fractions 1, 5, and 9 from four lanes across 2 cartridges were run on a 1D gel to demonstrate the gel-to-gel and cartridge-to-cartridge reproducibility of the system.

Programmable Methods for Targeted Isolation

The Gelfree 8100 Fractionation System is the first product to provide user-programmable molecular weight-based protein purification with liquid phase recovery. Simple adjustment of the fraction collection intervals using the touch screen interface allows isolation of analytes at a targeted molecular weight in a single fraction with near quantitative recovery.

To develop a method for targeted protein isolation using the Gelfree system, the user samples the elution window at high frequency over the expected mass range. The peak width is plotted and the fraction collection interval defined. For all subsequent runs, the optimized fraction collection interval is used to isolate and purify the target analyte in a single fraction. Fraction collection windows for a given analyte depend on the amount of total protein loaded.

Programmable Protein Isolation at Varying Protein Load. Bovine serum albumin loaded into the Mid Mass Gelfree 8100 cartridge and collected per standard procedures. Peak width varies between 2 and 7 minutes, FWHM. Width increases at higher loading. Fractions were collected at 90 second intervals to define the elution window. Using this elution time data, collection times were optimized for maximum recovery. The gel image on the right shows the Gelfree fraction (GF) containing albumin, 1 μg and 25 μg, compared to control.

G8100_Manual_v1-4.pdf (753,457kb)

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Gelfree® 8100 Fractionation System