Tutor profile: Noor H.
If you are purifying a His-tagged protein and you run an SDS-PAGE of your final purified sample following nickel chromatography and size-exclusion chromatography, what are some reasons you could obtain a smear, or more than one band?
Nickel chromatography separates proteins based on their affinity to nickel. The His-tagged protein is designed to interact more strongly with the nickel column than average proteins, and so will elute in a different fraction. However, some proteins can have non-specific interactions with the nickel column, and thus small amounts of other protein could elute with your desired protein in the same fraction. This could potentially contaminate your sample and result in additional bands in your SDS-PAGE. However, size-exclusion chromatography following nickel chromatography is designed to minimize this occurrence, because size-exclusion chromatography further separates proteins based upon a different criterion: their size. Each fraction collected with contain a protein within a given size range, and thus a fraction can be collected of your desired protein at its expected size. This further reduces the chances of other proteins co-eluting with your desired protein, although if you happen to have a protein that interacts non-specifically with your nickel column and is of similar size as your desired protein, it may still contaminate your sample and result in additional bands in your SDS-PAGE. Alternate reasons aside from contamination that could explain a smear or multiple bands include the following. Your desired protein sample may be undergoing self-cleavage or degradation before you load it onto the SDS-PAGE, which would result in multiple bands or a small smear. Alternatively, your protein could be modified through glycosylation, methylation, or some other process, which would affect the molecular weight of the protein and lead to multiple bands or large smears.
Why is DNA replication mutagenic? What evolutionary purpose could there be to such a trait, and why have DNA polymerases not evolved to have 100% fidelity?
DNA replication is mutagenic because the DNA polymerase does not have perfect fidelity, and misincorporates bases at a certain frequency. Although there is a risk of deleterious substitutions being made, mutations are also necessary for evolution to take place, and thus a certain basal rate of mutagenesis is beneficial to an organismal population as a whole, as it lends the opportunity to the acquisition of new traits that may improve the fitness of individual organisms. If DNA polymerases had evolved to have 100% fidelity, evolution in these organisms would occur at a much slower rate, and they would be outcompeted by organisms with more promiscuous DNA polymerases.
In Bram Stoker's Dracula, the titular character is a creature that inspires fear and abhorrence. What is it about the vampire's nature that is so threatening to the characters, and how does it relate to Victorian fears of the time?
One aspect of the threat Dracula poses to the cast of predominantly English characters is his otherness, and specifically, the othering influence he threatens to exert on them. Lucy's near-transformation into a sensual vampire is a very clear illustration of the palpable effect Dracula has, changing her from a demure Victorian lady to an object of sexual desire. In a time when female sexuality was a subject of anxiety, Dracula serves as an exploration of that fear, and the ramifications of sexual liberation perpetrated by a non-English influence.
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