Affinity Chromatography Lab Report
Affinity chromatography technique is used to separate proteins found in a mixture of solution. Affinity chromatography uses the strong interaction between a given protein and its corresponding molecule. In today’s lab, affinity chromatography was used to purify L-lactate dehydrogenase, which contains histidine-tagged protein. The histidine- tagged protein forms a strong interaction with the Ni-NTA column due to the presence of nickel ions. Varying concentration of imidazole was added to the column in order to elute out the histidine-tagged protein. Imidazole competes with the histidine side chains to bind with the nickel ions which thus, elute out the desired protein. Three different concentrations of imidazole 25mM, 250mM, and 400mM were
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This means that there are not as many imidazole bonded to the nickel ions as the histidine. SDS-Page separates molecules based on molecular weight therefore; this once again corresponds with the results. Since, the elution of 25mM imidazole produced a higher molecular weight it thus shows that the protein is still bonded to the nickel ion disputed through the small Rf …show more content…
Again with a greater Rf value shows that the protein has traveled further down on the SDS Page and this means that all of the imidazole is bonded to the nickel ions, which cause all of the proteins to be eluted out of the column. Experimentally determined molecular weight of tagged LDH is 42 kilodalton and the theoretical molecular weight of tagged LDH is 37 kilodalton due to the great similarities between the two results show that our gel had run well.
Our tagged LDH was successfully purified due to the similarities in the experimental and the theoretical molecular weight. When LDH is eluted with the various imidazole solutions you are exchanging the binding between nickel ions and LDH to nickel ions and imidazole. By increasing the concentration of imidazole you are increasing the amount of imidazole in the solution thus; giving it more chances to over come the bond between the nickel ions and LDH.
A possible source of error may be that LDH may have not fully dissolved in the column before the addition of the
This means that there are not as many imidazole bonded to the nickel ions as the histidine. SDS-Page separates molecules based on molecular weight therefore; this once again corresponds with the results. Since, the elution of 25mM imidazole produced a higher molecular weight it thus shows that the protein is still bonded to the nickel ion disputed through the small Rf …show more content…
Again with a greater Rf value shows that the protein has traveled further down on the SDS Page and this means that all of the imidazole is bonded to the nickel ions, which cause all of the proteins to be eluted out of the column. Experimentally determined molecular weight of tagged LDH is 42 kilodalton and the theoretical molecular weight of tagged LDH is 37 kilodalton due to the great similarities between the two results show that our gel had run well.
Our tagged LDH was successfully purified due to the similarities in the experimental and the theoretical molecular weight. When LDH is eluted with the various imidazole solutions you are exchanging the binding between nickel ions and LDH to nickel ions and imidazole. By increasing the concentration of imidazole you are increasing the amount of imidazole in the solution thus; giving it more chances to over come the bond between the nickel ions and LDH.
A possible source of error may be that LDH may have not fully dissolved in the column before the addition of the