Thermo Scientific HisPur Cobalt Superflow Agarose is a tetradentate-chelating Superflow 6 support charged with divalent cobalt, designed for high-purity, large-scale purification of polyhistidine-tagged proteins.
HisPur Cobalt Superflow Agarose is a highly crosslinked, durable resin that does not compress at flow rates used for medium- to large-scale FPLC purifications. The resin holds up well to a variety of chemicals and pH values, and is compatible with common clean-in-place procedures. Compared to nickel and other ligands commonly used for immobilized metal affinity chromatography (IMAC), cobalt provides greater specificity for His-tagged protein purification. The specific binding of HisPur Cobalt Superflow Agarose results in purer elution fractions, reducing the need for extra polishing steps.
- Robust – highly crosslinked 6% agarose Superflow beaded support tolerates linear flow rates up to 1200cm/hour with minimal leaching of cobalt due to a tetradentate chelator
- High yields – binds 20mg of 6xHis-GFP per mL of resin at a linear flow rate of 150cm/hour, can bind greater than 30mg per mL of resin depending on flow rate
- High purity – specificity of cobalt binding to histidine-tag generally results in elution fractions with less than 10% contamination of non-specific proteins
- Compatible – maintains function after exposure to a wide variety of chemicals and pH values
- Cost effective – resin is stable through multiple cycles of cleaning and reuse
- Large scale FPLC purification of polyhistidine tagged proteins
Properties of Thermo Scientific HisPur Cobalt Superflow Agarose.
||Superflow 6 Resin, highly crosslinked 6% agarose
||60 to 160μm
||Recommended: 150cm/hour (binding, wash, elution)
||Metal: ≥11μmol Co2+/mL resin
||Static: approx. 30mg 6xHis-GFP/mL resin
Dynamic‡: approx. 20mg 6xHis-GFP/mL resin
||1M acetic acid pH 2, 1% SDS pH 7, 6M guanidine-HCl, 70% ethanol for ≥1 week at 37°C; 8M urea, 10mM DTT, 5mM TCEP for ≥2 hours at 22°C
||pH 3 to 9 for ≥1 week at 4°C
pH 2 to 3 or 9 to 12 for ≥ 2 hours at 22°C
||Up to 25 times
|† Maximum Linear Flow Rate Conditions: Column Dimensions (w x h): 13mm x 38mm (5mL resin); Sample: deionized water at room temperature; Linear Flow Rate: volumetric flow rate (mL/min) x 60 (min/hr)/cross-sectional area (cm2).
‡ Dynamic Binding Conditions (10% breakthrough): Column Dimensions (w x h): 5mm x 50mm (1mL resin); Sample: 1mg/mL 6xHis-GFP (27kDa) pure protein in 20mM NaH2PO4, 300mM NaCl, 5mM imidazole; Flow Rate: 1mL/min
Affinity chromatography is often used as a quick and easy approach for the purification of recombinant proteins. One such method of purification involves the binding of a polyhistidine tag to a divalent metal cation that has been coordinated by a chelator immobilized on a beaded support. For immobilized metal affinity chromatography (IMAC) purification of His-tagged proteins, the type of bead, chelator, and metal immobilized influences purity, yield, and flow rate performance of these purifications.
The highly crosslinked 6% agarose Superflow beaded support tolerates larger linear flow rates than agarose resins that have lower amounts of crosslinking. The HisPur Cobalt Superflow Agarose protocol utilizes gentle wash and elution conditions and typically produces greater than 90% pure target protein. Protein purities achieved with HisPur Cobalt Superflow Agarose are generally higher than those achieved with nickel IMAC resins making HisPur Cobalt Superflow Agarose a valuable tool for users interested in one-step purifications. Additionally, HisPur Cobalt Superflow Agarose is perfect for larger scale purifications that require the resin to withstand medium pressures without compressing.
|Medium scale FPLC purification of 6xHis-GFP produces >90% purity of target protein. 100g of biomass containing overexpressed 6xHis-GFP was lysed with 1L of lysis buffer supplemented with Halt Protease Inhibitor (Part No. 78439) and loaded onto an equilibrated 200mL column (50mm x ~100mm) of Thermo Scientific HisPur Cobalt Superflow Agarose or Clontech™ TALON™ Superflow (Takara Bio Co.) at a flow rate of 20mL/minute, then washed until baseline with wash buffer containing 15mM imidazole. Bound protein was eluted with buffer containing 150mM imidazole. Fractions containing purified 6xHis-GFP were pooled and quantitated using Pierce 660nm Protein Assay (Part No. 22662). Load, flow-through, wash, and eluate fractions (5μg) were separated by SDS-PAGE, stained with coomassie dye and evaluated using myImageAnalysis Software (Part No. 62237) to determine purity. Total yield, recovery, and purity were nearly identical for both resins.
Review of His-tagged fusion protein production and purification
Other Affinity Resins for His-tagged Protein Purification
Glutathione Resins for GST-tagged Protein Purification
B-PER Bacterial Protein Extraction Reagent
Protease Inhibitor Solutions and Tablets
Slide-A-Lyzer Dialysis Flasks (250mL capacity)