BCHS 3304 Lecture Notes - Lecture 10: Molecular Mass, Isoelectric Point, Sodium Dodecyl Sulfate
8 Oct 2016
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Protein purification: step 1: find a good source of protein. Tissues may have specific proteins so be aware of this: step 2: get the protein out of cells. Hypotonic shock= water rushes into the cell. Break it mechanically with a blender or something: step 3: maintain stability. Prevent denaturation= protein loses active shape/ conformation. Protect against protease, inhibitor, change in ph: step 4: assay the protein. Quantify how much protein you have as you purify is. The assay must be performed at each step of the purification process. Use antibodies that are specific to the target protein: this will separate the protein mixture as only the target will bind to the antibody. Add a second assayable enzyme: can view thru color change, or fluorescence. Types of assay have an assayable portion: direct= use 1 antibody, indirect= use of 2 antibodies, capture= use of 3 antibodies, step 5: determine concentration of protein. Bradford assay= use it to calculate concentration of protein.
