BIOL 2020 Lecture Notes - Lecture 7: Isoelectric Point, Elution, Hemoglobin
29 Apr 2016
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Start: protein of interest, source material, assay for detection of protein in various samples. Salt fractionation ( salting out with (nh4)2so4: separates by solubility. Gel filtration/ molecular exclusion: separates by size. Af nity chromatography: separates by af nity to a ligand. Binding is dependent on pi of protein and ph (charge changes with ph) Proteins have a net positive charge at phs below their pi. Proteins have a net negative charge at phs above their pi. Proteins are neutral at ph equal to their pi. In example above, protein a will have a negative charge and protein will have a positive charge at ph 6: both proteins will have a negative charge at ph 8. Changing ph can also be used to elute bound proteins from an ion exchange column. Igg has 4 subunits (2 heavy chains, 2 light chains) If they are the same size (same point) the intensity will be greater.
