BIOL 301 Lecture Notes - Lecture 9: Tandem Affinity Purification, Fusion Protein, Two-Hybrid Screening
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Ph for which the protein is neutrally charged. So low ph has more protons to counter more negative proteins. Separate protein based on mw: smaller proteins travel further. Buffer with lots of carrier anions (gly- or cl-) Not agarose, but polyacrylamide (pa) gel matrix (cid:1) polymers of acrylamide linked by bis-acrylamide linkers (cid:1) 5-15% pa. More concentration = tighter sieve, take longer for proteins to move thru gel, used to study smaller proteins. Agarose pores are too big, proteins are smaller than dna or rna so it would flow thru too fast. Vs southern/ northern gel and blots: can"t separate just based on charge, bc proteins have different shapes and charges too. Method: need to apply sds and linearize the proteins (denature them), which is ok bc we no longer care abt protein activity. Boil proteins, this is to destroy any ppi into single subunit. Use beta mercaptol: reducing agent to break s-s bonds within proteins.
