1. Methods of Protein Purification

Jun 26, 2020 by

Thousands of proteins are found in cells. All are made up of sequences of only 20 amino acids, although biologically they have exceptionally different functions, chemically they are very similar.

However, if a biochemist is interested in studying the characteristics and function of a single protein, chances are he will have to purify it from all the others.

This, Until very a few years ago, it was a very big challenge, since the separation and purification of compounds by traditional chemical methods implied methodologies to distinguish physical and/or chemical differences between one substance and another to be able to separate them.

In today’s world, laboratories have very strong and robust methods for protein purification. In the past time required to purify the protein was about several months or years, but now it can be done in days.

The purpose of this article is to explore some of the most widely used methods to purify the protein.

The work of purification of a compound, found within a tissue or organ, begins with some method of maceration, to break the cells and leave the content free, suspended in some liquid, a buffer is generally used for this purpose. This is called a homogenate.

In the container containing the homogenate, all the compounds found in this form in the cytoplasm of the cell are in solution and the intracellular organelles such as mitochondria, lysosomes, or nuclei and fragments of membranes are suspended. Homogenate is a very complex mixture, so the next step is to make a partial separation of its components.

  • Centrifugation Method

The centrifugation of the homogenate allows us to separate in a quite acceptable way the constituent components.

The principle of this procedure is to increase the force of gravity g, using a centrifuge whose rotor rotates at many revolutions per minute.

Under these conditions, the heavier particles go to the bottom of the container faster than the heavier ones.

In a homogenate subjected to the described conditions, it is possible to separate, for example, the nuclei from the mitochondria, since the former are heavier than the latter, so a fraction consisting of nuclei while the mitochondria will remain in suspension.

After several centrifugations, there are several fractions, which can be either rich in nuclei, or mitochondria, or ribosomes, etc.

If, for example, the protein to be studied is in the mitochondria, the other fractions can be discarded and only the one that is rich in these organelles can be conserved, to work on subsequent purification steps with only this part.

  • Dialysis Process

The next step is to separate all the proteins from other existing molecules in the mixture, such as metals, carbohydrates, or lipids, the method is called the dialysis process.

Dialysis uses a pouch that has pores small enough to prevent large molecules of proteins from passing through, but large enough for small molecules to pass through.

Some fraction of the homogenate is placed inside the bag and it is hermetically sealed. The system thus constructed is placed in a container with distilled water, which is continuously renewed.

Under these conditions, there is a difference in concentrations between the liquid inside the bag and the distilled water.

There is going to be a tendency to equalize concentrations, so the molecules inside are going to try to get out into the distilled water, but since the protein molecules can’t go through the pores, the transport of materials will continue until the concentrations in the two compartments are equal.

If the distilled water is changed continuously, it will prevent it from reaching equilibrium, so those small molecules will continue to come out.

If this process continues, only protein molecules will remain inside the bag. With this procedure, you now have a mixture of many proteins so the next step is to purify what is required.

Since proteins are electrically charged molecules and this varies according to the pH of the solution in which they are dissolved, a very common method of achieving their separation is utilizing the electrophoresis technique.

  • Gel Filtration Method

Another way to separate proteins of different sizes is by gel filtration.

In this method, a hydrophilic gel is used, made with a polymer, such as dextran, which is placed in a tube.

This material has the property of forming a molecular mesh in which the pores are very small. A solution containing the protein mixture is pipetted into one end of the tube.

The smallest proteins can pass through the pores of the gel and the largest cannot, in such a way that large proteins come out of the other side of the tube, the small ones will be delayed because they have to cross the entire molecular mesh.

If the liquid that is coming out of the column is collected in different containers, these contain different types of protein.

  • Chromatographic Methods

In chromatographic methods, the solution of protein is flown through a column, packed with various materials. Different proteins behave differently with materials in the column.

2.Size Exclusion Chromatography

In this method, smaller molecules have to travel a larger volume in a porous matrix. The protein of a certain size will need a variable amount of solvent before being collected at the end of the column.

3.Ion Exchange Chromatography

In this method, compounds are separated based on ionic charge. The column is selected on the base of the strength of the charge.

Anion is used to extract the negatives charged compounds whereas cations are used to separate the positively charged molecules.

4.Affinity Chromatography

Affinity chromatography is a type of liquid chromatography that is based on the reversible binding between the analyte of interest and a specific ligand, immobilized on an inert solid support.


High-pressure liquid chromatography is a separation technique based on the different retention of the components of a sample dissolved in the mobile phase when passing through the stationary phase.

It is characterized by the high operating pressure necessary to achieve the optimum speed of the mobile phase.

The interaction between the solute of the mobile phase and the stationary phase can be modified by choosing different solvents and stationary phases, consequently being a highly versatile technique.


By combining several of the methods described here, it is possible to obtain a completely pure protein in a test tube. Having achieved this, you are ready to study it, regarding its structure and function.

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