Immobilized metal affinity chromatography (IMAC) purification of secreted histidine‐tagged proteins in eukaryotic cell culture supernatants is often problematic. Incompatibility with the cell culture media appears as stripping of the immobilized metal ions required for binding of target proteins. Due to low target protein concentration in cell culture supernatants, large sample volumes are often needed, aggravating the stripping effect. In this study, purifications of histidine‐tagged proteins were performed using two novel Sepharose™ based IMAC media; Ni Sepharose excel and magnetic His Mag Sepharose excel. Both media have a new type of chelating ligand with exceptionally strong binding of nickel ions. Data showing successful purification of histidine‐tagged proteins from CHO cell culture supernatants will be presented. Furthermore, the characteristics of new media enabled purification of target protein from insect cell culture supernatants. The purification was easily scaled up from 20 μl His Mag Sepharose excel beads to 1 mL pre‐packed columns.

The histidine‐tag is commonly used to facilitate purification of recombinant proteins. For immobilized metal‐ion affinity chromatography (IMAC) the most used metal ion is nickel. However, also cobalt has been shown useful obtaining high purity with good yield of his‐tagged proteins in IMAC applications.We have designed three formats containing cobalt IMAC media, convenient for rapid and simple small‐scale purification and screening of his‐tagged proteins. The formats, a gravity flow column, a spin column and a 96‐well plate, all allow efficient parallel purification of proteins in μg to mg scale within 40 minutes using fast and easy protocols to help ensure pure target proteins. His‐tagged green fluorescent protein expressed in E. coli was used as model protein to demonstrate the functionality of the formats. The results show that recovery and purity is > 80 % using all formats. The repeatability has proven to be high for all formats with < 10 % difference from average for each run. A comparison between cobalt and nickel IMAC media was performed and our products containing cobalt IMAC media show higher purity compared to products containing nickel IMAC media while still maintaining a high yield.

The binding of streptavidin to biotin is one of the strongest known non-covalent biological interactions and hence a powerful tool used in affinity chromatography. Biomolecules can easily be fused with biotins, to which immobilized streptavidin ligands on chromatography matrices can bind. The usage of magnetic beads for affinity based purifications simplifies small-scale purifications and provides high flexibility with scales ranging from μl to ml. In this study, we show that the magnetic bead medium Streptavidin Mag Sepharose™ offers leading performance concerning binding capacity and achieved purity. Binding capacity of Streptavidin Mag Sepharose for biotinylated rabbit monoclonal IgG was 1.7 mg/ml bead slurry. Immunoprecipitation of 7.5 μg/ml transferrin in a background of 5 mg/ml E.coli protein showed a 420-fold enrichment. Scaling up immuno-precipitation experiments 10 times or changing sample concentration 100 times, resulted in equal purity and recovery (%). The characteristics of the Mag Sepharose beads are; simplify handling by reducing the risk of beads sticking on tubes and pipette tips. Moreover, the beads respond quickly to magnetic fields resulting in rapid separation of the beads from the sample, completed within seconds.

There are many different types of post‐translational modifications (PTM) identified till now of which protein phosphorylation is one of the most important in eukaryotic cells and plays an essential role for regulating various cellular processes.Immobilized Metal Affinity Chromatography (IMAC) is a common technique used for purification and enrichment of phosphopeptides and is based on the affinity of the metal ion for the phosphate group. Phos SpinTrap™ Fe is a new sample preparation kit based on Fe Sepharose™ 6 Fast Flow. The kit is designed for single use small‐scale phosphopeptide enrichment. A mixture of different phosphorylated proteins was spiked into a background of E. coli extract. The protein mixture was cleaved with trypsin and purified on a Phos SpinTrap Fe column. Several phosphopeptides were detected with masspectrometric methods after the enrichment. Conclusion: The Phos SpinTrap Fe kit shows high specificity for phosphopeptides and can be used for enrichment of phosphopeptides from complex samples.