Until the beginning of the 20th century, typical separation technologies included precipitation, filtration, and crystallization.

Since the advent of modern liquid chromatography, it is rare that any one of these early techniques is used alone. Today, a combination of techniques is generally used to assure the quantity, quality, and desired purity of the protein target.

A number of protein precipitation methods are frequently used today. Protein precipitation with a neutral salt, such as ammonium sulfate, is useful for purification and enrichment of a target protein from an extract without loss of activity.

At industrial scale, Cohn’s cold ethanol fractionation/precipitation methods from the 1940s are still used to produce human serum albumin, immunoglobulins, and other blood-based products. However, chromatography methods, in combination with ultrafiltration, have started to replace Cohn fractionation since they offer higher purity and yield, and are easier to automate. Today most purification schemes involve some form of chromatography. As a result

chromatography has become an essential tool in every laboratory where protein purification is needed.

In affinity ultrafiltration, the protein to be purified is complexed with a macroligand composed of a soluble polymer or an insoluble microparticle with covalently bound, target protein-specific affinity ligands. The complex is trapped by an ultrafiltration membrane, whereas unwanted proteins pass through the membrane. The unwanted proteins are removed from the system by the carrier liquid. The system is then supplemented with an agent eluting the target protein by dissociating it from the microligand complex. The purified protein then passes the membrane, while the macroligand is trapped by it. The macroligand can be re-used after regeneration. Affinity ultrafiltration has a number of advantages over other protein purification techniques: 1) commercial availability of ultrafiltration systems with various high-productivity designs; 2) availability of presynthesized macroligands, which can be supplemented with additional, easily manufactured, commercial latex-based macroligands; 3) rapid separation of large solution volumes; 4) repeated use of equipment, enabling consecutive purification of different proteins; 5) simple scale-up and automation procedures.

Until the beginning of the 20th century, typical separation technologies included precipitation, filtration, and crystallization.

Since the advent of modern liquid chromatography, it is rare that any one of these early techniques is used alone. Today, a combination of techniques is generally used to assure the quantity, quality, and desired purity of the protein target.

A number of protein precipitation methods are frequently used today. Protein precipitation with a neutral salt, such as ammonium sulfate, is useful for purification and enrichment of a target protein from an extract without loss of activity.

At industrial scale, Cohn’s cold ethanol fractionation/precipitation methods from the 1940s are still used to produce human serum albumin, immunoglobulins, and other blood-based products. However, chromatography methods, in combination with ultrafiltration, have started to replace Cohn fractionation since they offer higher purity and yield, and are easier to automate. Today most purification schemes involve some form of chromatography. As a result

chromatography has become an essential tool in every laboratory where protein purification is needed.

In affinity ultrafiltration, the protein to be purified is complexed with a macroligand composed of a soluble polymer or an insoluble microparticle with covalently bound, target protein-specific affinity ligands. The complex is trapped by an ultrafiltration membrane, whereas unwanted proteins pass through the membrane. The unwanted proteins are removed from the system by the carrier liquid. The system is then supplemented with an agent eluting the target protein by dissociating it from the microligand complex. The purified protein then passes the membrane, while the macroligand is trapped by it. The macroligand can be re-used after regeneration. Affinity ultrafiltration has a number of advantages over other protein purification techniques: 1) commercial availability of ultrafiltration systems with various high-productivity designs; 2) availability of presynthesized macroligands, which can be supplemented with additional, easily manufactured, commercial latex-based macroligands; 3) rapid separation of large solution volumes; 4) repeated use of equipment, enabling consecutive purification of different proteins; 5) simple scale-up and automation procedures.

The most preferable method to get pure protein: 1- Ultra filtration.

2- Fractional precipitation with organic solvent .

3- column chromatography (ion exchange and gel filteration)

all these methods with arrangement give one band of protein on SDS gel electrophoresis