Protein Analysis

a) 2290 b) 2698

1) Tissue homogenisation by sonication, blending, pestle and mortar 2) Separisation of the released material by centrifugation 3) Several chromotography steps 4) Confirmation of protein purify by electrophoresis 5) Comfirmation of protein identift by western immunoblotting 6) Confirmation of protein identity by mass-spectrometry

Velocity sedimentation Equlibrium sedimentation

Subcellular components sediment at different speeds according to their shape and size when layered over dilute solution containing sucrose After centrifugation the different components can be collected individually by puncturing the plastic centrifuge tube and collecting drops from the bottom

Subcellular components move up or down when centrifuged in a gradient until they reach a position where their density matches their surroundings This is due to a sucrose gradient The bonds can also be collected by putting a hole in the tube

Porous beads are used where the pores are big enough for the right protein to enter the beads Larger molecules flow through the solvent and smaller molecules are ‘retarded’

Mixture of proteins are passed through the columns The columns have different matrixes so proteins can be separated according to charge, hydrophobicity, size or their ability to bind to other molecules

Takes advantage of biologically important binding interactions on protein surfaces The substrate is bound to a matrix It is used to purify proteins

Short DNA oligonucleotides of a specific design can be immobilised in this way and used to purify the DNA binding proteins

Increasing salt concentrations are used to compete for the ionic interactions between the proteins and beads Proteins are then eluted from the column

Carboxy methyl CM

diethylaminoethyl (DEAE)

Proteins with the lowest affinity for the ion exchange resin as it passes directly through the column

sodium dodecyl sulphate polyacrylamide gel elctrophoresis

An electric field is applied to a solution so proteins are separated according to their net charge The solution contains SDS which binds to hydrophobic regions of protein molecules causing them to unfold

Breaks S-S linkages in proteins

Where the proteins are separated by both charge and size

In proteonomics

A stable pH gradient

It has no net charge and no longer migrates in the electric field

A detectable enzyme or a fluorescent dye

a) Electrophoresis transfer b) Incubate with antibodies (AB) c) Wash off the antibodies d) Antibody detection e) Incubate with enzyme linked antibody (AB2) f) Wash off excess enzyme linked antibodies g) Add substrate to detect activity h) Enzyme detection

Rabbit or mouse antibodies are injected into a donkey The donkey antibodies against the other antibodies can then be purified and labelled

Trypsin