Protein purification - created from Mind Map

Página 1

Choose biological maaterial

Preparation

Purification

Protein detection

Obtain initial extract

Techniques<br><br><ul><li>To breal the cells and get the proteins out.</li></ul>

Differential centrifugation<br><br><ul><li>Separate proteins from other nonsoluble particles based on difference of mass/density</li></ul>

dif. osmotic pressure

ultrasonic waves

detergents

organic solvents

french press

Homogenator

Blender

supernatant with proteins

stabilize the protein

Methods

pH-neutral

T not exceed 60/65ºC

prevent air contact/foam, causes oxidation

protases-protease inhibitors

contaminants-antimicrobial agents

usually on ice4ºC

Solubility<br><br><ul><li>Based on the precipirtation of proteins</li></ul>

Molecular weight

Electric charge

Polarity

Specific binding

Salting out

pI precipitation

precipitation with organic solvents<br><br><ul><li>Act by making the solution less polar, they lower the dielectric cte of solutions There are less water molecules available to solvate proteins.</li></ul>

Ethanol

Acetone

Dialysis

Ultracentrifugation

size exclution cromatography

Electrophoresis SDS gels

polyacrylamide sulphate catalyst

acrylmide

N,N-methylenebisacrylamide

Ion-exchange chromatography

Electrophoresis 2D

Isoelectric focusing

SDS electroph

Isoelectric focusing

ampholytes-gradient pH

Adsortion chromatography

Reverse phase chromatography

Hydrophobic interaction chromatography

liquid-liquid partition

protein is denatured

hydrophilic stationary phase with hydrophobic groups covalently

molecules bind by weak hydrophobic interactions

native structure of protein is maintained

Affinity chromatography

ELISA

Immunoblotting/Wetern blotting

Enzyme-linked immunosrbent assay