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.

Gel filtration (GF) is an excellent tool to acquire information about sizing (identity), purity, and the multimeric state of a protein of interest. Superdex 200 and Superdex 75 are outstanding GF media for such analysis. To speed up analysis and keep sample and buffer consumption at minimum, two prepacked short GF columns have been developed, Superdex 200 5/150 GL and Superdex 75 5/150 GL. With lengths of only 15 cm and volume of 3 ml, these columns allow rapid analysis (6-12 min/run) with minimal sample (4-50microl) and buffer consumption.

The Strep(II)‐tag is an ideal tag due to the small size (8 aa, 1 kDa), often making removal of the tag unnecessary. The prepacked StrepTrap™ HP 1 ml & 5 ml columns have been specially developed for high specificity towards the Strep(II)‐tag facilitating fast and convenient purification. Purification is done under physiological conditions and mild elution preserves the activity of the target protein. The 34 um bead size of the resin allows elution in narrow peaks, minimizing the need for further concentration steps. In this work dual‐tagged fluorescent protein, (His)6‐mCherry‐Strep(II) expressed in E. coli, was purified in a purification procedure including two affinity columns, StrepTrap HP followed by HisTrap™ HP. The second affinity column was necessary for removal of a truncated variant of the target protein. The multi‐step purification was performed fully automatically using ÄKTAxpress™. Another dual‐tagged protein expressed in insect cells was purified to high purity in a single step using StrepTrap HP. The final purity was above 95% according to SDS‐PAGE. Finally, six repeated purifications of a Strep(II)‐tagged protein expressed in E. coli were performed on the same StrepTrap HP column with 0.5 M NaOH regeneration between the runs. The final purity and yield of the target protein remained almost unchanged, no tendency of decreasing values, demonstrating the high reproducibility.

The success of protein production and subsequent analysis are dependent on the expression levels, solubility and purification of the protein. Maltose Binding Protein (MBP) is often used as an affinity fusion tag. Several studies have shown that MBP‐tagged proteins have high solubility compared to other tagged proteins.The new MBPTrap™ HP consists of an affinity medium, Dextrin Sepharose™ High Performance, packed in 1‐ml and 5‐ml HiTrap™ columns. The medium has high specificity and capacity. Additionally, it can withstand harsh Cleaning In Place (CIP) procedures, like sodium hydroxide, without losing binding capacity. The HiTrap format enables automated protocols to be used on ÄKTAdesign™ systems as well as with syringe or laboratory pump.In this work, MBP‐tagged proteins from E. coli were purified on prepacked 1‐ml or 5‐ml MBPTrap HP columns. The purity of the eluted target proteins was greater than 90 %. A stability study was performed, six purification runs each followed by a CIP run (0.5 M NaOH) were made on the same column. The results show no significant change in yield or purity. The results of an automated two step purification on ÄKTAxpress™ is shown. The affinity chromatography step on MBPTrap HP was followed by a gel filtration step.In summary, the results show that the prepacked MBPTrap HP column has high binding capacity and the eluted target protein is very pure. Furthermore, the medium is stable in 0.5 M NaOH.

The histidine-tag has become the most used affinity tag to facilitate the purification and detection of recombinant proteins. Immobilized metal ion affinity chromatography (IMAC) with Ni2+ is the standard method for purifying histidine-tagged proteins. His GraviTrapTM is a new pre-packed column, filled with 1 ml Ni SepharoseTM 6 Fast Flow, for simple gravity-flow purifications of histidine-tagged proteins from clarified or unclarified samples. In this work we have used His GraviTrap to purify different histidine-tagged proteins from E.coli lysates. Clarified lysate containing approximately 40 mg of a histidine-tagged protein was applied and a recovery close to 100 % was obtained in only 40 minutes. His GraviTrap and a comparable product from another supplier were compared for purifying a histidine-tagged protein during native conditions. Results show that the total purification time was approximately five-times faster on His GraviTrap with similar purity in the eluted pools. Purification of a high molecular weight (Histidine)10-tagged protein from a clarified E.coli lysate on His GraviTrap was performed in 25 minutes. Analysis of the eluate on SDS-PAGE showed however a lot of bands. Western blot analsysis using an anti-His antibody was performed, and it was shown that the “impurities” are truncated versions of the target protein.