BIOC 2580 Lecture Notes - Lecture 4: Sodium Hydroxide, Differential Centrifugation, Significant Figures
9 Jan 2017
School
Department
Course
Professor
Document Summary
A protein sample is placed in an ultracentrifuge spinning at 10,000 to 500,000 x gravity (g) At these g-forces, molecules sediment 9move down the tube) at a rate that depends on size and shape. By (cid:373)easuri(cid:374)g sedi(cid:373)e(cid:374)tatio(cid:374) velo(cid:272)ity, it"s possi(cid:271)le to (cid:272)al(cid:272)ulate the (cid:373)ole(cid:272)ular (cid:373)ass of a protei(cid:374) Electrophoresis: separation based on movement of charged molecules in an electric field. Rate of movement depends on size, shape and charge. To prevent movement of the solution, the separation is carried out in a porous gel. Polyacrylamide gel is easily prepared in lab, had right porosity for proteins 10 kda - 1000 kda. A typical gel is 5015% polymer and 90-95% water with conductive buffer. In sds-polyacrylamide gel electrophoresis (sds-page) the protein is pre-treated with the detergent sodium dodecyl sulfate (sds) When sds is used, native protein charge is swamped out, and the overall negative charge is used to move the protein through the gel (towards the + electrode)
