MBIO 2420 Lecture Notes - Lecture 9: Polyclonal Antibodies, Lysis Buffer, Western Blot
7 Dec 2016
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How to determine lysis efficiency: quantitate amount of protein in lysate vs. cell/tissue debris (western blot, spin the sample - has supernatant - solubilize portion. Also have a pellet - not solubilized: then use western blot to test for amount of protein. Purpose: to remove proteins that may non-specifically bind to protein a/g or beads: good for chip, not necessarily ip, beads are with protein a/g and some non protein of interest may attach to the beads. Reduce background in later steps: 1hr rotating at 4 degrees. Secondary reagents - use wide mouth tips: protein a - agarose beads. Bacterial fc receptors from staphylococcus aureus: protein g - agarose beads. Bacterial fc receptors from group g streptococci. Spin it - so beads and non protein of interest is at the bottom. Wash beads separately in lysis buffer prior to use.
