Metal Ions

A search of the literature on IMAC reveals a bewildering array of metal ions that have been used in this technique (e.g. Ag +, Al3 +, Ca2 +, Co2 +, Cr3 +, Cu2 +, Eu3+, Fe3 + , Hg2+, La3 + , Mn2+, Nd3+, Ni2+, Yb3+, Zn3+). The reason for this is that the nature of the metal ion (and indeed its chelator) influences the selectivity and affinity of the protein interaction. The most commonly used metals can be grouped into the 'hard' and 'soft' types - reflecting their electron orbital configuration and ability to act as electron acceptors. In free solution the metal ions exist with a shell of water molecules. Upon chelator or protein binding the water is displaced and a coordination bond to the metal ion is formed by the donation of free electron pairs from atoms in the chelator or in the amino acids (e.g. N, O and potentially S) of the protein. As such the atoms behave as monodentate ligands with the affinity estimated to be in the micromolar range. Both the protein and the immobilized chelator have the potential to be polydentate. For protein binding the 'soft' metal ions (e.g. Cu2+, Co2+, Zn2+, Ni2+ ) show a preference for coordination with nitrogen-containing functional groups such as the imidazole of histidine (either ยง or s nitrogens). The 'hard' metal ions (e.g. Al3+, Ca2+, Fe3+) show a preference for oxygen-containing groups such as carboxyls or phosphates found in phosphorylated proteins. These preferences are exploited in the nature and types of proteins purified with particular combinations of chelator and metal ion, and, to a certain extent, with the choice of buffer conditions. Within the 'soft' metal group a rank order of affinity for histidine residues has been established. In increasing strength of binding this order is Co2+ k Zn2+ <Ni2+ < Cu2+. Histidine is relatively rare, representing only 2.2% of the amino acids across all proteins with many containing none or none accessible on their surface. This provides a built-in selectivity for certain native proteins. The use of genetic engineering to introduce a 6His tag further exploits the selectivity for histidine. The preferred use of Ni2 + in IMAC with 6His tagged proteins is in part due to its higher coordination number (Cu2+ =4, Ni2+ = 6) and the fact that the weaker binding potential of the Ni2# is compensated for by the tag, thus providing an even greater degree of selectivity over other proteins from the recombinant host.

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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