Protein Separation & Purification Techniques
Applications of Separation of proteins ( purified proteins):
Quantitative analysis
Therapeutic / clinical use
Research purpose
Separation of proteins from other proteins and molecules is achieved by combination of several methods , based on Properties such as :
Solubility
Molecular size
Molecular charge
Specific binding of the protein to the specific substance
Principle of Dialysis : Small molecules ,such as salts can be removed from protein solution by dialysis using semipermeable membrane tube/bag.
Process of Dialysis : it is performed by adding/ passing protein salt solution to a semipermeable membrane tube/bag (cellophane bag). When tube is immersed in a dilute buffer solution ,small molecules will pass through and large molecules can be retained in the tube, depending on the pore size of the dialysis membrane .
Biochemical Application of Dialysis : for removal of ammonium sulphate solution ( desalting)during protein purification
Clinical Application of Dialysis : management of kidney failure patients
Principle of Gel filtration : the rate at which a molecule flows through the column is dependent on its size and shape. Small molecules enter the pores of the gel molecule but larger molecules cannot. Therefore, large molecules take a more direct route through the column . The volume of solvent available for these is greater than that for the lager molecules. So the small molecules flows through the column more slowly.
Gel filtration / molecular sieve chromatography/ molecular chromatography: is a technique used to separate solutes based on their molecular size .
The sample solution containing a mixture of proteins is passed through a column of polymeric material previously equilibrated with the buffer.
Gels used to prepare column : cross- linked dextran (e.g. Sephadex), Agarose (e.g. Sepharose), polyacrylamide , polystyrene
Proteins of different molecular size penetrate into the internal pores of the gel to varying degrees .
Large Protein molecules that cannot enter the pores remain in the excluded volume , whereas small Protein molecules enter the pores. Protein of intermediate size are excluded based on their size.
Ultracentrifugation for Separation of proteins based on molecular size : High speed centrifugation can separate a protein solution in various components . The rate at which a protein can sediment in a centrifugal field depends on its :
Size
Shape
Molecular weight
For proteins of similar shape, centrifugal force sediments those molecules having large molecular weight .
Centrifugal force is expressed in terms of sedimentation coefficient (S) = v/w2r
Used for separation of Proteins/peptides ,amino acids, carbohydrates
Purification of proteins ,enzymes , immunoglobulins , membrane receptors , nucleic acids
Determination of molecular weight of a protein
Analysis of hormones , brain amines ,vitamins, drugs
Qualitative and quantitative analysis of complex mixtures
In Clinical laboratory for the identification amino acids (amino acidurias), peptides/proteins, sugars ,drugs in serum or urine or biological fluids
Ion-exchange chromatography: separation is carried out in the column (consist of a long tube) filled with Ion exchange resin carrying either anionic or cationic groups.
Anion exchange resins : have fixed positive charge groups and exchangeable anions e.g. diethylaminoethyl (DEAE) Sephadex
Cation exchange resins : have fixed negative charge groups and exchangeable cations e.g. (Dowex 50), carboxymethyl dextran(CM Sephadex), CM(carboxymethyl) cellulose . Strong Cation exchangers are used for separation of amino acids (amino acid analyzers).
The affinity of each peptide /protein molecule for the resin is affected by
pH, which determines the ionization state of the protein molecule.
Principle of Affinity chromatography : is a technique for separating proteins / macromolecules based on the specific affinity (non covalent binding) of a molecule for its ligand (such as an enzyme for its substrate/ cofactor or protein for its antibody) which is attached to gel matrix .
Technique of Affinity chromatography :
The sample (mixture)to be separated is applied to a column of ligand bound to a matrix such as crossed-linked dextran or agarose (Sepharose).
The molecule that has high affinity for the ligand binds to it ,while other molecules are eluted out of column. The immobilized ligand acts as molecular fishhooks to selectively pick up the desired protein while the remaining proteins pass through the column.
The bound molecule is eluted by changing the conditions (change in pH or salt concentration) so that binding /affinity is does not exist(break protein-ligand interaction) .