Question

List, discuss, and evaluate the major technique used in separating proteins, including: solubility, size, dialysis, SDS-PAGE, charge-ion exchange chromatography, size and charge 2D gels, specific ligand binding affinity chromatography, antigen-antibody recognition (western blot, ELISA); mass spectrometry of peptide and proteomics

Answer 1

Answer:

Purpose of separation:

The separation or purification step is essential to separate protein and non- protein parts of mixtures. The following techniques used to separate the desired protein from the mixture.

Based on Size:

Based on the size the proteins are used to separate from a mixture by using porous gels. The basic principle is that smaller molecules have to transverse a larger volume in a porous matrix.

Based on the solubility :

Proteins are precipitated out from aqueous solutions when added salt concentration exceeds a critical level is known as salting out. Because the salts bound to the water and there is no water to hydrate the proteins.

1. Salting out process :The separation of the proteins based o solubility occurs in two steps:

1. Salt is added at a concentration just below necessary to precipitate out the protein of interest. The solution is centrifuged to remove if any less soluble protein is present.

2. Then the concentration of salt is increased at a point just above the precipitation of the proteins occurs of interest.

3. Then the desired precipitated proteins are removed by centrifugation.

2. Isoelectric precipitation pI: pI of a protein is the pH where the net charge on the protein is zero. It separates based on the different isoelectric point of their different amino acid sequences ( relative numbers of anonic and cationic groups) by adjusting pH of a solution. When the pH is adjusted to the pI of a particular protein it precipaitates leaving the other proteins in solution.

3. Solvent fractionation: This separation is based on solubility of a protein depends on the dielectric constant of the solution that surrounds it because this alters the magnitude of the electrostatic interactions between the charged groups. As the dielectric constant of the solution decreases, the magnitude of the electrostatic interactions between the charged increases.

4. Denaturation of contaminating proteins: Many proteins are separated by this by adjusting pHs acidic or basic. The proteins which are stable at high temperatures are separated by this technique. Contaminating proteins are separated by precipitating out while the proteins of interest remains in the solution.

Proteomics which explains the study and characterization of complete set of proteins present in a organ / organism, cell at a given time.

Charge and size 2D electrophoresis gel technique:

This is the separation process of complex protein mixtures based on

Molecular charge present in the first dimension (pI value).

Mass / size present in the second dimension (molecular weight).

It also provides information regarding molecular weight, quantity, pI, possible post translational modification.

SDS- PAGE is a separation or purification electrophoresis technique:

This technique allows separation of proteins by mass or molecular weight. In this matrix or medium is a poly acrylamide (PA) - based discontinuous gel. In this SDS (sodium dodecyl sulfate) about 1.4 grams is used, which binds to a gram of protein, i.e., one SDS molecule per two amino acids is used.

Procedure for both Charge and size 2D electrophoresis gel technique & SDS- PAGE:

1. Separation of proteins based on pI value, which indicates decreasing pI value.

2. This gel is then soaked in SDS solution which is fitting it on an SDS PA gel.

3. Separation of proteins based on molecular mass, which indicates decreasing molecular mass.

Specific ligand binding affinity chromatography, which uses unique aspects of the biological / individual chemical structure of a protein to affect its separation. By taking an suitable interacting ligand, which is having high natural specificity to the target protein there by it results in highly selective separations can be achieved.

Western blot technique separates the protein mixture based on molecular weight, type through gel electrophoresis. These results are then transferred to membrane by providing band for each protein.

ELISA (Enzyme – linked immuno-sorbent assay) which is a plate based assay technique, detects and separates by quantifying peptides, proteins etc. In this, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme.

Mass spectroscopy:

This technique is the best choice of a given proteomic separation technique, which is gel free or gel based.

It is the primary tool for protein separation which involves in 3 steps:

1. Protein ionization and generation of gas phase ions.

2. Separation of ions according to their mass to charge ratio and

3. Detection of ions.

Thank you.........