Lab skills 2 | Bright Minds Mentori

Centrifugation

Separate substances by differing densities

less dense, soluble & kept

Successive increases in speed/time in centrifuge result

in increasingly less dense substances being spun down into pellet.

more dense, insoluble & removed

Chromatography

1. Paper/Thin Layer

Separating amino acids/sugars based

on their relative SOLUBILITY in a solvent.

2. Affinity

Specific molecules in gel beads are used to separate a soluble mixture of protein.

1. Non target proteins

Have low affinity, washed out column.

2. Target protein

Has high affinity and binds to molecules

in beads to separate from rest of mixture.

Gel Electrophoresis

Charged macromolecules (protein/DNA) move through an

electric current applied to a gel matrix

1. Native Gel Electrophoresis 2. SDS Page Gel Electrophoresis

Undenatured proteins separated Denatured proteins have

based on uniform negative charge & move to +ve electrode by

- size - size only - smaller move

- shape further

- charge

3. Isoelectric Focusing

Separating proteins using their different Iso electric points.

Isoelectric points

When a protein has no net charge & precipitates out of solutiom

Soluble proteins separated by their isoelectric points using an

electric current with a gel matrix containing a pH gradient

using a series of buffers.

Buffers

chemicals which hold pH constant despite addition of acid/alkali.