LIFESCI 3 Lecture Notes - Lecture 6: Column Chromatography, Affinity Chromatography, Northern Blot
Document Summary
Rna isolated from different tissues and put the same amount of rna from each tissue into different wells. Take sample cells, grind them up and isolate proteins from cells -> pour into column. Matrix has negatively charged beads -> positively charged proteins stick onto beads. Negatively charged proteins come out of the column. First fraction of proteins coming out of the column are most negatively charged (repelled) Add high salt solution to the beads -> disrupt ionic interactions between proteins & beads -> collect positively charged proteins: gel filtration (size exclusion) chromatography. La(cid:396)ge p(cid:396)otei(cid:374)s (cid:272)a(cid:374)"t get t(cid:396)apped (cid:271)(cid:455) (cid:271)eads, so (cid:272)o(cid:373)e out f(cid:396)o(cid:373) (cid:272)olu(cid:373)(cid:374) fi(cid:396)st. Beads with pores in them are used. The next fraction contains medium sized proteins. Small proteins enter aqueous spaces within beads and are the last ones to exit. Collect fractions of what come out of the column.
