Proteins exist in tissues or cells as complex mixtures and each type of cell contains thousands of different proteins. However, there is no single method can remove any type of protein from the complex mixture until now. Here we would like to share with you the current popular methods of protein separation.

Method of Separating Proteins According to Their Solubility

01 Salting Out of Protein

The salt saturation required by different proteins for salting out is different, so the target protein can be salted out with precipitation by adjusting the salt concentration. These proteins remain their natural property and can be dissolved again without denaturation. Neutral salt (generally for ammonium sulfate, magnesium sulfate, sodium sulfate, sodium chloride, sodium phosphate. Ammonium sulfate, as the most used salt, has the advantage of small temperature coefficient and large solubility, also not easy to induce degeneration) has a significant effect on protein solubility, generally, its solubility increases with rising salt concentration at low salt concentration, this is regarded as salt bath; When the salt concentration continues to rise, the solubility of protein precipitation falls in varying degrees successively, this phenomenon is called salting out. Add a lot of salt to the protein solution salt ions of high concentrations (such as ammonium sulfate SO4 and NH4) have strong force of hydration and can seize hydration layer of protein molecules, so the protein colloidal particle coagulate and precipitate due to loss of water.

Factors Affecting Salting Out Include:

Temperature: This can generally be done at room temperature, except at low temperatures (4 degrees) for temperature-sensitive proteins. Generally, protein solubility decreases at low temperature, but some proteins (e.g., hemoglobin, myoglobin, albumin) are less soluble at higher temperatures (25 degrees) than at 0 degree, and are more likely to be salted out.

pH: Most proteins have the lowest solubility in concentrated salt solutions at isoelectric points.

Protein concentration: When protein concentration is high, proteins to be separated are often precipitate with other proteins (co-precipitation phenomenon). Therefore, the serum should be diluted with the same amount of normal saline before salting out to keep protein content at 2.5-3.0%.

After proteins are separated by salting out and precipitation, the salt in the protein needs to be removed. The commonly used method is dialysis, that is, putting the protein solution into a dialysis bag (commonly use cellophane), dialysis with buffer solution and constantly replace buffer solution. Dialysis takes a long time, therefore, it is better to carry out at low temperature. In addition, making dextran gel Tandex G-25F or Tandex G-50 through column can also be used to remove salt, which takes relatively short time.

02 Isoelectric Point Precipitation Method

When the protein is in static state, the electrostatic repulsion force between particles is the smallest, so is the solubility. The isoelectric point of various proteins is different, adjusting pH of the solution can reach the isoelectric point of a protein to precipitate it. However, this method is rarely used alone, it can be used with salting-out method.

03 Low Temperature Crganic Solvent Precipitation Method

Most proteins can be salted out by decreasing solubility with the organic solvent, methanol, ethanol or acetone which can be miscible with water. The resolution of this method is higher than that of salting out, but denaturation is prone to occur for proteins, so that this method should be done at low temperature.

Method of Separating Proteins According to Their Molecular Size

01 Dialysis and Ultrafiltration

Dialysis uses semi-permeable membranes to separate proteins of different molecular sizes.

Ultrafiltration uses high pressure or centrifugal force to coerce water and other small solute molecules through the semi-permeable membrane, while proteins remain on the membrane. Different pore sizes can be selected to retain proteins with different molecular weights.

02 Gel Filtration Method

Also known as molecular exclusion chromatography or molecular sieve chromatography, this is one of the most effective methods of separating protein mixtures according to the size of the molecules. The most commonly used packing materials in the column are dextran gel (Tandex series) agarose gel (Tanrose series) and agarose gel (SuperTandex pg series).

Methods of Separating Proteins According to Their Charged Properties

01  Electrophoresis

Proteins can be separated by their mobility in the electric field due to their molecular weight and amount of charge at the same pH condition. Isoelectric focused electrophoresis uses ampholyte as the carrier, ampholyte forms pH gradient which gradually increases from the positive pole to the negative of pole, when a certain charged proteins electrophoresis there, then stop after arriving at their isoelectric point position of the pH. This method can be used for analysis and preparation of a variety of proteins.

02 Ion Exchange Chromatography

Ion exchange reagent can be divided into cation exchange reagent (such as: SP Tanrose 6 FF) and anion exchange reagent Tanrose 6 FF (Q), when protein solution flows through ion exchange column chromatography, proteins with opposite charge of ion exchange reagent adsorb on the ion exchange reagent, then proteins can be eluted by changing pH or ionic strength.

Separation Method According to the Ligand Specificity – Affinity chromatography

03 Affinity Chromatography

An extremely efficient method for separating proteins is to separate on the basis of some proteins bind with ligand, specifically but not covalently. It often takes only one step to separate protein to be purified from a complex protein mixture with high purity.

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