Affinity Chromatography

Reactive chlorotriazinyl dyes are easily and safely immobilized on agarose and other polyhydroxylic supports (e.g. cellulose, polyacrylates) or, less often, on amino-group-bearing supports, after nucleophilic substitution in alkaline environment. Alternatively, a diaminoalkane (e.g. 1,6-diaminohexane) spacer molecule can be chemically attached to the chloro-triazine ring of the dye, before the resulting conjugate is immobilized on various pre-activated polyhy-droxylic supports. Although chemical immobilization of reactive dyes on polyhydroxylic supports is the most straightforward and widely used technique, perhaps the most unusual one is immobilization via adsorption. For example, it is possible, first, to chemically attach to the reactive dye a hydrophobic 1H,1H-pentadecafluorooctylamine tail by nuc-leophilic substitution. Then, the dye-tail conjugate can be physically adsorbed on to hydrophobic fluorocarbon particles. Hydrophobic fluorocarbon supports can be transformed into surface-hydrophilic materials. In this case, hydrophobic perfluorocarbon tails are chemically attached to hydrophilic poly-vinylalcohol (PVA). Afterwards, the PVA-tail conjugate is physically adsorbed on to the hydrophobic surface of TeflonĀ® particles. Therefore, the transformed TeflonĀ® surface is hydrophilic and rich in hydroxyl groups through which reactive dyes can be immobilized.

At the end of the immobilization reaction, the coloured adsorbent is washed to remove free dye, packed in a chromatography column, and equilibrated with the appropriate buffer for protein purification. The sample is applied and nonadsorbed proteins are washed off, before the desired protein is desorbed (eluted) by changing the composition of the buffer eluent. This is realized either by nonspecific desorption techniques (i.e. change of the ionic strength or pH) or by specific desorption techniques, for example, introducing competing substances (i.e. substrates, inhibitors, metal chelators) in order to selectively perturb the enzyme-dye complex on the column.

In the development of a protein purification protocol, some form of adsorbent screening exercise should be performed as the first step. The adsorbent exhibiting the highest purification factor and recovery for the targeted protein on the one hand, and on the other hand the adsorbent showing no adsorption of the protein of interest but good total protein adsorption, are promising candidates for employment in positive and negative affinity chromatography, respectively.

Although beaded porous polyhydroxylic supports have been used for decades in biochromatography, rapid mass transfer has only been achieved recently thanks to two novel chromatography supports. The 'flow-through particles', which are exploited in perfusion chromatography, have pores wide enough (0.6-0.8 |m) to allow convective flow through them. Smaller diffusive pores along the throughpore channels of these particles provide a high adsorption area with diffusion path lengths less than 1 |im. These particles reduce process time by improving mass transfer. An alternative for the same purpose is offered by hyperdiffusion chromatogra-phy; this uses rigid particles with their entire pore volume filled by a homogeneous flexible soft hydrogel where the affinity ligands are immobilized. The proteins can diffuse freely through the hydrogel network and interact with the ligand.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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