Data using the cartridge format for automated liquid chromatography (LC) systems
A variety of desalting and affinity-purification supports are available pre-packed in 1mL and 5mL Thermo Scientific Pierce Chromatography Cartridges for processing manually or by automated liquid chromatography (LC) systems. The cartridges attach directly to ÄKTA™ or FPLC Systems without additional connectors. Cartridge products include an accessory pack (also available separately) of tubing fittings and Luer-Lok™ fittings that provide compatibility with the other popular LC systems and manual syringe processing. Cartridges can be used individually or connected in series to obtain even greater column capacity (Table 1; Figure 1).
Table 1. Pierce Chromatography Cartridge properties
Recommended and maximum flow rates are general; values differ slightly for individual products.
|Feature||1mL Cartridge||5mL Cartridge|
|Dimensions||0.7 x 2.7cm||1.3 x 3.8cm|
|Desalting flow rate
|0.2 to 1mL/minute
|1 to 5mL/minute
|Affinity flow rate
|0.1 to 1mL/minute
|0.5 to 2mL/minute
|Maximum pressure||0.3 mPa (43 psi or 3 bar)||0.3 mPa (43 psi or 3 bar)|
Figure 1. Scale diagram of Pierce Chromatography Columns. Viewed at 72dpi, this drawing is essentially actual size.
This article summarizes performance data (in the form of chromatograms and stained gels of resulting fractions) for selected desalting and affinity columns in the Pierce Chromatography Cartridges format. These include cartridges containing Thermo Scientific Zeba Desalting Resin (7K MWCO), Pierce Protein A Plus Agarose, Pierce Protein G Agarose, Melon Gel Support, HisPur Cobalt Resin and HisPur Ni-NTA Agarose. Other resins are also available as Pierce Chromatography Cartridges, including Pierce Glutathione Agarose (see Related Products links in side panel).
Zeba Desalting Chromatography Cartridges, 7K MWCO contain a proprietary high-performance gel-filtration resin that has exceptional desalting and protein-recovery characteristics compared to other commercially available resins (Figure 1). Even very dilute (25µg/mL) protein samples can be successfully processed to obtain greater than 95% retention (removal) of salts and other small molecules (<1000Da) and good recovery of proteins and other macromolecules (>7000Da). The cartridges provide fast, easy and reproducible chromatographic separations. They can be regenerated for multiple uses and efficiently process samples from 50 to 1500µL.
Protein A cartridges
Thermo Scientific Pierce Protein A Plus Agarose consists of purified native Protein A that has been covalently immobilized at high density onto high-quality crosslinked 6% beaded agarose (CL-6B). Pierce Chromatography Cartridges are just one of many sizes and types of columns and kits in which this affinity resin is available.
Figure 3. Comparable antibody yield and purity achieved with Protein A Chromatography Cartridge. Normal human serum (60mg) was applied in PBS to a 1mL Thermo Scientific Pierce Protein A Cartridge (Part No. 89924) (A) and a 1mL GE Healthcare HiTrap HP Column (B) and eluted with 0.1M glycine, pH 2.8, using a flow rate of 1mL/minute. The arrow denotes the start of the low-pH elution. The yield of human IgG was 6.85mg and 6.88mg, respectively. Fractions were separated by SDS-PAGE and the gels were stained with Imperial Protein Stain (Part No. 24615). M = MW marker, L = sample load, FT = flow-through and E = elution.
Protein G cartridge
Thermo Scientific Pierce Protein G Agarose consists of purified recombinant Protein G that has been covalently immobilized at high density onto high-quality crosslinked 6% beaded agarose (CL-6B). Pierce Chromatography Cartridges are just one of many sizes and types of columns and kits in which this affinity resin is available.
Figure 4. Comparable antibody yield and purity achieved with Protein G Chromatography Cartridge. Normal human serum (60mg) was applied in PBS to a 1mL Thermo Scientific Pierce Protein G Cartridge (Part No. 89926) (A) and a 1mL GE Healthcare HiTrap Column (B) and eluted with 0.1M glycine, pH 2.8, using a flow rate of 1mL/minute. The arrow denotes the start of the low pH elution. Fractions were separated by SDS-PAGE and the gels were stained with Imperial Protein Stain (Part No. 24615). M = MW marker, L = sample load, FT = flow-through and Elution = elution fractions.
Melon gel cartridge
Thermo Scientific Melon Gel IgG purification resins and kits enable purification of antibodies from serum and other sample types. Melon Gel Resin binds and removes serum proteins, thereby allowing pure IgG to be collected in the flow-through fraction. The procedure is gentle because no harsh elution conditions are involved. Packages and kits are available to perform purification using gravity-flow columns, spin columns or chromatography cartridges.
HisPur cobalt cartridge
Thermo Scientific HisPur Cobalt Resin products use a tetradentate chelating agarose support that is charged with divalent cobalt (Co2+) to enable specific, fast and gentle purification of histidine-tagged fusion proteins from a variety of sample types. Compared to Ni-IDA and tetradentate Ni2+ chelate resins, Co2+ resin binds histidine-tagged proteins with higher specificity (less off-target binding) and releases them with gentler conditions (lower concentrations of imidazole).
Figure 6. Purification of 6xHis-GFP from E. coli lysate using a HisPur Cobalt Cartridge. His-tagged green fluorescent protein (GFP) was extracted from E. coli using Thermo Scientific B-PER Bacterial Protein Extraction Reagent in Phosphate Buffer (Part No. 78266) containing Halt Protease Inhibitor Cocktail, EDTA-Free. The lysate was diluted 1:1 with equilibration/wash buffer (50mM sodium phosphate, 300mM sodium chloride, 10mM imidazole, pH 7.4) and applied to a 1mL HisPur Cobalt Chromatography Cartridge (Part No. 90093) at a flow rate of 0.3mL/min. The cartridge was washed with equilibration/wash buffer until the baseline absorbance at A280 was reached. His-tagged GFP was eluted (50mM sodium phosphate, 300mM sodium chloride, 150mM imidazole; pH 7.4) and selected fractions were analyzed by SDS-PAGE and Thermo Scientific GelCode Blue Stain Reagent (Part No. 24592). M = MW Marker; S = non-fractionated lysate; FT = flow-through.
HisPur Ni-NTA cartridge
Thermo Scientific HisPur Ni-NTA Resin is a nickel-IMAC resin for routine affinity purification of His-tagged fusion proteins. The specially prepared support consists of beaded agarose derivatized with the nitrilotriacetic acid (NTA) chelation moiety and loaded with divalent nickel ions (Ni2+). The immobilized metal affinity chromatography (IMAC) resin provides exceptional binding capacity and performance for recombinant His-tagged protein purification.
The experiments reported here were conducted in 2007, except for the HisPur Ni-NTA experiment (Figure 7), which was done in 2010. All chromatography runs were performed using an ÄKTA* System (GE Healthcare).
Thermo Scientific Zeba Spin Desalting Columns, 7K MWCO, 75µL are polypropylene devices containing a proprietary high-performance size-exclusion chromatography resin that provides an excellent protein desalting and recovery in a centrifuge format.
Features of Zeba Spin Desalting Columns, 7K MWCO, 75µL:
- High recovery—low-binding resin maximizes protein recovery
- Fast—no fraction screening or waiting for protein to emerge by gravity-flow
- Easy-to-use—no cumbersome column preparation or equilibration
- Flexible—available in spin columns, filter spin plates and cartridges for a range of needs
- Economical—great performance at lower cost than other commercially available cartridges
Even very dilute (25 µg/mL) protein samples can be successfully processed to obtain greater than 95% retention (removal) of salts and other small molecules (< 1000 MW) and good recovery of proteins and other macromolecules (> 7000 MW).
Learn more about Thermo Scientific Zeba Spin Desalting Columns
For Research Use Only. Not for use in diagnostic procedures.