Chromatography cartridges for desalting and affinity purification
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.
||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
||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).
RESULTS and DISCUSSION:
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.
Figure 2. Efficient salt removal and protein recovery with Desalting Chromatography Cartridge. Bovine serum albumin (1mg) in 1M NaCl was applied to 5mL Thermo Scientific Zeba Desalting Cartridge (Part No. 89935) (A) at a flow rate of 5mL/minute. Cartridge profile shows isocratic elution of BSA (blue) and NaCl detected by conductivity (brown). Greater than 95% of the BSA was recovered and more than 95% of the was salt removed. Results for the Thermo Scientific Pierce Cartridge (A) were essentially identical to those obtained with the more expensive GE Healthcare (B) and Bio-Rad (C) products.
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 acheived 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 acheived 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.
Figure 5. Effective reverse-affinity purfication of antibody with Melon Gel Cartridges. Normal human serum (1mL) was applied to a 1mL Thermo Scientific Melon Gel Cartridge (Part No. 89932) at a flow rate of 1mL/minute. Protein contaminants bind to the resin while the antibodies flow through (FT). Volume of recovered antibody = 4 to 5mL. Regeneration (R) solution strips the bound protein contaminants so that the cartridge can be reused multiple times. The purity of the isolated antibody was evaluated by SDS-PAGE (right panel) and stained with GelCode Blue Stain Reagent (Part No. 24590). M = MW Marker, L = sample loaded, FT = flow-through and R = regeneration fractions.
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 (Part No. 78415). 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.
Figure 7. Purification of 6xHis-GFP from E. coli lysate using a HisPur Nickel-NTA Cartridge. Bacterial lysate (130mg total protein) containing over-expressed 6xHis-GFP (green fluorescent protein) was diluted 1:1 with equilibration buffer and applied to a 1mL HisPur Ni-NTA Chromatography Cartridge (Part No. 90098) at a flow rate of 1mL/min. The cartridge was washed with PBS, 68mM imidazole until the baseline absorbance was reached. The 6xHis-GFP was eluted with PBS, 300mM imidazole. Elution was monitored at 280nm (blue line; total protein) and 485nm (red line; GFP-specific). Selected fractions were analyzed by SDS-PAGE. Gel lanes were normalized to equivalent volume. M = MW marker, L = lysate load, FT = flow-through and E = elution.
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).