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MIA

Protein Purification

WooHoo
Gel filtration chromatography (size) big molecules have a shorted path because they cannot fit trough, can only go around. medium is a bit longer. small is longest, can fit through so must go through all.
Dialysis Semi-permeable membrane (excludes material at molecular level), Permeable (allows small molecules and water to pass), Impermeable (does not allow large molecules).
Dialysis for rapid dialysis, maximise area of membrane/volume of solution.
SDS-page Similar to electrophoresis but SDSP added to sample which applies a negative charge to each protein in proportion to its mass. SDS allows proteins to be separated strictly by molecular weight.
Silver stain used to make invisible protein bands visible (BLACK)
Comassie blue makes protein bands blue, less sensitive.
Mass spectrometry Accurate reading of molecular weight, down to a single atom.
Cation-exchange chromatography Has -vely charged polymer beads. Rate of movement determined by net charge at the pH used. More -ve proteins move faster because like charges repel.
Isoelectric focussing IEF Charge on a protein is affected by pH. A pH gradient is set up and an electric current is passed through. When protein reached pI, it stops.
2D-PAGE first dimension = IEF, separation by charge. second dimension = SDS-PAGE, separation by size. decreasing Mr vertically, decreasing pI horizontally.
Created by: nyna
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