Protein purification. Scientist in laboratory
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Protein purification: 4 techniques used by researchers

We can define protein purification as isolating a single type of protein from a biological tissue or microbial culture. To isolate many proteins, one cannot use the same method for all of them. So, we must always keep the goal in mind when choosing the purification method to achieve the desired concentration. 

Various peptide synthesis companies offer different high-quality peptide products, from automated peptide synthesizers to high-quality reagents and laboratory equipment. But have you ever wondered what techniques they use for protein purification? Purifying proteins is a crucial first step to understanding their function. The methods used for protein purification include:

1. Chromatography

2. Centrifugation

3. Protein electrophoresis

4. Cell disruption

1. Protein purification through chromatography

Researchers can purify proteins using various chromatographic techniques. Some of these methods include:

Ion exchange chromatography (IEXC)

The method involves the separation of proteins based on their net charge. You may prepare columns to facilitate both anion exchange and cation exchange. A prevalent protein purification technique, the IEXC method has a high protein binding capacity, is relatively easy to perform, and effectively removes unwanted impurities in the sample. 

Chromatography based on hydrophobicity

The process of hydrophobic interaction chromatography (HIC) and reverse-phase chromatography (RPC) works on this principle. 

With HIC, we achieve the separation of proteins by treating the column with a highly ionic buffer to allow the hydrophilic proteins to bind to the column. RPC reduces the polarity of the mobile phase by adding organic solvents.

Gel filtration or size-exclusion chromatography

With this method, you can separate larger proteins from smaller proteins by using a small volume of the eluate. Since the stationary phase does not interact with the solutes, it exhibits excellent sensitivity and does not lead to sample loss.

Affinity chromatography

As the purest chromatography technique, affinity chromatography completes the purification process of proteins. Under favorable conditions, different proteins will interact precisely with specific ligands, allowing the absorption of the target from the extract as it passes through the column.

2. Protein purification by centrifugation

Once we break down large portions of tissue, centrifugation is an excellent way to remove them. Often, different centrifugation methods are necessary for obtaining protein extracts. Centrifugation time and rate determine whether we draw subcellular organelles into pellets. 

Using a similar technique, you can separate proteins from supernatants. Three-dimensional structure plays a crucial role in protein interactions and functions. Through hydrogen bonds, ionic bonds, and Van der Waals forces, amino acid side chains provide the chemical pockets required for these functions. If they interrupt these interactions with a buffer high in salt or low in pH, they can then collect the proteins with another round of centrifugation. 

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3. Protein purification through protein electrophoresis

Electrophoretic separation of protein components in a sample is a technique commonly used in proteomics. This technique facilitates separating different protein types by passing them through an electrical current through a polyacrylamide sieving gel matrix. Electrophoresis can separate and detect protein components using various methods. These include non-denaturing PAGE, SDS-PAGE, tris tricine, bis tris, and isoelectric focusing (IEF).

Protein Electrophoresis Techniques and Applications

Native PAGE 

This technique allows for separating proteins by mass and charge while maintaining the proteinsā€™ structure and biological activity. 

It’s an excellent method for separating proteins in their native state, but it may separate patterns that can affect the outcome of your research.

SDS-PAGE

It works by degrading the sampleā€™s proteins by using sodium dodecyl sulfate (SDS). SDS forms non-covalent bonds with protein components to impart an overall negative charge and change its complex tertiary shape into a rod-like extension. 

This technique allows proteins to move between gel matrix layers according to size and provides a faster way to determine a protein’s molecular weight.

Tris Tricine

A typical application of this technique is to separate small proteins and peptides that have a degree of molecular weight of less than ten kDa. Various buffers are available, including tricine sample buffer, tris-tricine, and tris-tricine-SDS.

Bis-Tri

It is suitable for the separation of small and medium-sized proteins under denaturing conditions. Using MES SDS or MOPS/SDS running buffers, we can get different separation ranges.

Isoelectric Focusing (IEF)

When separating proteins based on their isoelectric points, IEF is most effective. Although this technique is quite valuable independently, we often use it as the first dimension in two-dimensional pages.

4. Protein purification through cell disruption

Breaking open cells can take several forms. Lysis involves lowering the ionic strength of the medium cells. Cells can swell and burst because of this. A mild surfactant may enhance lysis. Many bacteria, yeasts, and plants with protein walls can withstand such osmotic shocks; however, more vigorous disruption techniques are usually necessary.

It may be possible to degrade the cell walls with enzymes. For example, lysozyme is effective at destroying bacterial walls. Others include cellulose (from plants), glycanases, proteases, and mannans. A researcher can agitate tiny beads in suspensions of cells using mechanical means. During the high-speed bombardment of the cells, the beads break them open. An alternative method for lysing cells is to sonicate (using sound waves of 20-50 kHz). However, the technique is noisy and produces heat, which can be problematic for heat-sensitive compounds.

Conclusion

Protein purification involves several techniques mentioned above. Analyzing pure proteins enables us to determine amino acid sequences and to examine protein functions biochemically. The crystallization of proteins requires pure proteins, and we can study the tertiary structure by x-rays with an image of the functional protein.

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