OptioBioTM Glass Columns

We are happy to offer prepacked OptioBio™ glass columns designed for small-scale purification as well as screening and optimization in bioprocess development and scale-up. OptioBio 40S 10×100 and OptioBio 40Q 10×100 columns are prepacked with WorkBeads™ 40S and WorkBeads 40Q resins for ion exchange chromatography (IEC). The resins are designed for research and industrial scale purification of proteins, peptides and nucleic acids and utilise the difference in their surface charge. WorkBeads 40S is a strong cation exchange resin with sulfonate ligands, and WorkBeads 40Q is a strong anion exchange resin with quaternary amine ligands. The property of high-resolution separation in combination with low backpressure facilitates both capture and polishing purification applications.

• Prepacked for reliable and reproducible results
• Optimal for high-performance small-scale purification and method optimization in bioprocess development
• High throughput and purity

Affinity Chromatography

Affinity chromatography is a useful chromatography technique separating proteins in a reversible interaction between the target protein and ligand of bead. The interaction can be biospecific for example antibodies binding to protein A or non-biospecific for example histidine-tagged proteins binding to metal ions.

The chromatography technique provides high selectivity, high resolution and high capacity. High purity can often be achieved in a single step. Large sample volumes can be handled and samples can be applied under conditions that favor specific binding to the ligand. Elution is often performed under gentle conditions which helps preserve bioactivity. The target protein is eluted in a purified and concentrated form by modification of pH, ionic strength, or by introducing a competitive ligand.

Babybio Columns

BabyBio is our new range of ready-to-use mini columns available in 1 ml and 5 ml. Products are designed for quick, easy and convenient purification.

Column characteristics
The BabyBio column body is made from biocompatible polypropylene, which does not significantly interact with biomolecules. The top and bottom filters are made from polyethylene. These ready to use columns are delivered with plugs in the inlet and a snap-off end at the outlet. The columns can be connected a syringe, pump or chromatography system using fingertight fittings (coned 10-32) for 1/16” o.d. tubing.

Activated Resin

Resin for coupling your own ligands:

• Made from agarose, well established and well-known in the Biotechnology industry
• Simple coupling procedures at ambient temperature
• Stable at ambient temperature in aqueous solution and at neutral pH
• Suitable for coupling of ligands containing Sulphydryl,Amino- or Hydroxy- groups

Immobilized Metal Ion Affinity Chromatography (IMAC)

The first paper on immobilized metal ion affinity chromatography (IMAC) was published in 1975 by J. Porath et al. (1). The Uppsala research group showed that proteins can be separated on agarose beads with iminodiacetic acid (IDA) ligands (metal chelating groups) charged with divalent metal ions, e.g., copper (II) ions. The separation depends on the affinity of specific amino acid residues such as histidine, tryptophan and cysteine on the protein surfaces, to the immobilized metal ion. The technology was further improved by introducing the hexa-histidine affinity tag for recombinant proteins that made purification using IMAC very efficient. Other chelating groups, such as nitrilotriacetic acid (NTA) and tris(2-aminoethyl)amine (TREN), as well as different metal ions have been introduced later to generate improved selectivities. Frequently used metal ions are Ni2+, Co2+, Cu2+ or Zn2+. The bound proteins can be released either by lowering the pH or by introducing competitive ions such as imidazole or chelating agents such as EDTA.

Bio-Works offer IMAC purification media with chelating ligands based on NTA, IDA and TREN.  For generic purification of His-tagged proteins from, e.g., E. coli extracts we recommend using WorkBeads Ni-NTA. For samples from more difficult sources such as yeast or mammalian cells we recommend using the BabyBio His-tag Screening kits to evaluate most suitable combination of ligand and metal ion. For proteomics studies phosphopeptides and phosphoproteins can be separated on WorkBeads IDA medium charged with Fe3+ or Ga3+.

Reference
1. J. Porath et al., Nature 258, 598-599 (1975).

Kinesis Ltds’ promotional ad for BabyBio IMAC columns

 

Ion Exchange Chromatography (IEX)

In an aqueous environment most biomolecules are charged due to the presence of ionic groups within their structure. Depending on the pH of the environment these biomolecules carry a net positive charge, a net negative charge or no net charge. The pH value at which a biomolecule carries no net charge is called its isoelectric point (pI). The pI is highly specific for each type of biomolecule. When exposed to a pH below its pI a biomolecule will carry a positive charge.

It is therefore possible to separate out biomolecules according to their charge characteristics using a column of separation media that consists of beads covered in positively or negatively charged groups. A positively charged bead, known as an anionic exchanger, will tend to bind to biomolecules with a net negative charge, and a negatively charged bead, known as a cationic exchanger, will tend to bind to biomolecules with a net positive charge. The binding of the biomolecules to the beads is fully reversible and their removal (elution) is usually achieved through the flow of increasing amounts of sodium chloride salt down the column. The sodium or chloride ions compete with the binding of the biomolecules to the charged beads causing the biomolecules to be released and allowing them to be eluted out of the bottom of the column. The order in which the biomolecules are eluted is dependant upon their net charge; with the weakest charged coming off first.

Ion Exchange chromatography is a binding technique with very high binding capacities, high flow characteristics and potentially excellent resolution. It is therefore perfect for the separation of large volumes of sample (fluid feed) and fits well into the early or capture step of a purification methodology.