LIFESCI 3 Lecture Notes - Lecture 7: Affinity Chromatography, Fusion Protein, Expression Vector
Document Summary
Ls 3 - lecture 7 - molecular tools iii. Using fluorescent antibodies to visualize proteins within a dead cell (in-situ) Primary antibodies bind to the target (proteins) Can also see where different cellular proteins overlap. Fuse a sho(cid:396)t a(cid:373)i(cid:374)o a(cid:272)id se(cid:395)ue(cid:374)(cid:272)e that"s highly antigenic (his-tag) to terminus of protein sequence. His-tag does(cid:374)"t occur anywhere else in the body. Use the same strategy, fuse the sequence with gfp (green fluorescent protein) (isolated from some species, e. g. jellyfish) -> fusion protein with green light can produce gfp transgenic animals this way. (cid:271)ut: (cid:373)ake su(cid:396)e you do(cid:374)"t alte(cid:396) the p(cid:396)otei(cid:374)"s fu(cid:374)(cid:272)tio(cid:374) whe(cid:374) fused with gfp! Affinity chromatography: isolate proteins; does not disrupt any interactions. Immunofluorescence pinpoints location of proteins: movement & co-localization between proteins. Weste(cid:396)(cid:374) (cid:271)lot: dete(cid:272)t p(cid:396)otei(cid:374)"s p(cid:396)ese(cid:374)(cid:272)e; does not isolate p(cid:396)otei(cid:374)s (usually a follow up on chromatographic experiment to prove that you isolated the right proteins; purity of isolated fraction) (50 kda)
