MBIO 2420 Lecture Notes - Lecture 8: Lysis Buffer, Membrane Protein, Molecular Mass
7 Dec 2016
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Document Summary
Goal - to use gentlest conditions possible to solubilize protein and maintain: shape - antibody recognition, activity/function, protein-protein (dna) interactions. Purpose - to solubilize proteins and make accessible for antibody binding. Characteristics to consider about your protein of interest: expression profile - abundance, solubility - membrane vs. cytosolic vs. nuclear, activity/function, molecular weight. Smaller proteins are easier to solubilize: availability of specific antibodies, tagged protein-binding/activity/function. Has a small stretch of amino acids to it that adds an ab recognition site: sensitivity to mechanical disruption, machine to homogenize tissue may disrupt the protein. Factors to consider in a lysis buffer (lyses the cells, use for downstream washing): salt concentration. Stick around physiological salt concentration - 147 mm nacl. For nucleic acid - a bit basic so its soluble. Good for if you need the shape to say the same. Good if you just want to know the amino acid sequence. Ionic vs. non-ionic detergents: non-ionic - generally less denaturing.
