LIFESCI 3 Study Guide - Midterm Guide: Differential Centrifugation, Affinity Chromatography, Northern Blot
Document Summary
Physical disruption (differential or/and density gradient centrifugation) React with secondary antibody with fluorescent dyes. Fuse target proteins with fluorescent proteins/gfp: tag protein and express, make cell extract, precipitate with magnetic field or centrifuge depending on the immobilization of antibody. Smaller proteins can enter the pores in the beads so they flow through the column slower. Beads are covalently linked to antibodies or antigens. Protein recognized by the attached antibody/antigens on beads. All other proteins not recognized flow out first. Identify a specific protein: gel electrophoresis, transfer to membrane, wash twice with antibodies, detection. Detection of specific rna sequence: gel electrophoresis, transfer to membrane, probe with labeled dna, detection e. g. autoradiogram. Detection of specific dna sequence: gel electrophoresis. Detect specific dna sequences in the context of the cell of tissue: transfer to membrane, probe with labeled dna, detection e. g. autoradiogram. Count fluorescent dots (2 for each " normal) Heat dna strands to 95 to break h bonds.
