Separation Techniques of Protein

Separatory Techniques of Protein

• Precipitation is a reversible process.
• Types of techniques are-

1. Salt fractionation
2. Ultracentrifugation
3. Electrophoresis
4. Chromatography
5. Ultra filtration

 1. Salt fractionation–

• Salting In-  5% NaCl is used.
• Salting out- Excess Salt.
• Ammonium sulphate is most commonly used reagent for salting out in proteins.
• Sodium sulphate

 2. Chromatography-

• Chromatography is based on the principle of partition of the protein.
• The most non-polar amino acids migrate the farthest, due to their greater solubility in the organic solvent.

 a. Gel filtration chromatography or molecular sieving-

• Used for separation of molecules based on their size are Hydrophilic cross linked gels like agrose (Sepharose), dextran ( Sephadex)

b. Ion-exchange chromatography- Separation is made on the charge difference.

• The charged group commonly attached are

1.  diethylaminoethyl (DEAE) group- the negatively charged proteins associate with positively charged DEAE groups and replace chloride ions.
2. carboxylmethyl (CM) cellulase chromatography- negatively charged or neutral proteins have no affinity for CM, move faster.
3. High performance liquid chromatography (HPLC)-
4. Affinity chromatography– based on the high affinity of specific proteins for specific chemical groups called ligand. Specific protein molecules bind to specific ligand.
5. Hydrophobic interaction chromatography- hydrophobic ligand.

 3. Electrophoresis- isoelectric net charge is zero and do not move.

• Electrophoresis is the most common method of protein separation in the clinical laboratory.

Types of electrophoresis are-

1.  Polyacrylamide gel electrophoresis (PAGE) – on charge and molecular weight.
2. Sodium dodecyl sulphate (SDS)—PAGE- based on molecular size, molecular weight.
3.  Isoelectric focusing- on each protein has a different isoelectric point. 

Types of techniques are-

1. Salt fractionation
2. Ultracentrifugation
3. Electrophoresis
4. Chromatography
5. Ultra filtration

i. Salting In-  5% NaCl is used.

ii. Salting out- Excess Salt.

• Ammonium sulphate is most commonly used reagent for salting out in proteins.
• Chromatography- The most non-polar amino acids migrate the farthest, due to their greater solubility in the organic solvent. 
• Gel filtration chromatography or molecular sieving- Used for separation of molecules based on their size are Hydrophilic cross linked gels like agrose (Sepharose), dextran ( Sephadex)
• Ion-exchange chromatography- Separation is made on the charge difference.
• The charged group commonly attached are

1. diethylaminoethyl (DEAE) group- the negatively charged proteins associate with positively charged DEAE groups and replace chloride ions.
2. carboxylmethyl (CM) cellulase chromatography- negatively charged or neutral proteins have no affinity for CM, move faster.
3. Affinity chromatography- based on the high affinity of specific proteins for specific chemical groups called ligand. Specific protein molecules bind to specific ligand.
4. Hydrophobic interaction chromatography- hydrophobic ligand.
5. Electrophoresis- isoelectric net charge is zero and do not move.

• Electrophoresis is the most common method of protein separation in the clinical laboratory.
• Types of electrophoresis are-

1. Polyacrylamide gel electrophoresis (PAGE) – on charge and molecular weight.
2. Sodium dodecyl sulphate (SDS)—PAGE- based on molecular size, molecular weight.
3. Isoelectric focusing- on each protein has a different isoelectric point.