Your Guide to Protein Extraction: Overview, Uses, & More

Your Guide to Protein Extraction: Overview, Uses, & More

What Is Protein Extraction?

The only barrier to access the cellular content in animal cells is the plasma membrane (a lipid bilayer). However, in plants and bacterial cells, the cell wall provides an extra layer for the protection of cells.  

Therefore, for the isolation of the desired biomolecules, such as protein and nucleic acids, suitable extraction techniques are used. These techniques are based on the molecules to be extracted and are known as protein, DNA, and RNA extraction methods.  

Protein extraction is the process of isolation and purification of specific proteins from various biological samples, such as cells, tissues, and biological fluids, for successful proteomic experiments. It’s an essential first step for many analytical techniques.

Today, a variety of protein extraction techniques are available, all with a single goal of obtaining a high yield and purity of desired proteins. These techniques are chosen based on factors, such as:

  • Chemical and structural heterogeneity of proteins
  • Sample type or specimen source
  • Protein yield (depending on downstream applications)
  • The target protein’s cellular or subcellular location

Figure: Types of protein sources.

The first step of any protein extraction or cell fractionation method is cell lysis. To achieve high protein yield, mechanical and chemical techniques are used to homogenize cells or tissues containing proteins. The loss of protein due to enzymatic degradation is prevented by adding protease inhibitors to the lysate.  

In this article, we will review how protein extraction works, how it is performed, and its application in proteomics and biotechnology labs.

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How Does Protein Extraction Work?

Proteins are one of the major biomolecules of organisms. They govern an organism’s functions by regulating thousands of biochemical pathways. Therefore, these proteins can be used to learn about the biochemical pathways of diseases, the functioning of an organism, and gene expression through procedures like Gel Electrophoresis (such as SDS-PAGE), Western blot, Chromatography, Mass spectrometry, or ELISA.

Figure: The trend of increased purity of extracted protein with the progressive extraction and purification procedures.

The downstream applications are only possible after the extraction of proteins from tissue samples of interest using a suitable method.

The general steps involved in the process are as follows:

  1. Mechanically homogenize the tissue to break the cell membrane and release protein content or lyse them via sonication or enzymatic mechanisms.
  2. Filter the lysate to remove larger cell debris.
  3. Centrifuge the sample to remove cellular debris using differential centrifugation or density gradient centrifugation. Collect the supernatant containing cytoplasmic proteins, and discard the pellet.
  4. Resuspend the protein sample in a protein extraction buffer containing reagents such as salt, denaturant, reducing agents, and protease inhibitors. The detergent in the buffer solubilizes poorly soluble proteins, such as membrane protein, and the reducing agent prevents oxidative damage. The extraction buffer must be customized according to the extraction method used and the goal of the assay.  
  5. Perform biomagnetic separation of protein samples for the enrichment of protein of interest. These samples must be then purified using the techniques, such as protein purification chromatography and magnetic agarose beads.
  6. Measure total protein concentration using biochemical assays, such as the Lowry and Bradford assay, using a spectrometer at an absorbance of 280 nm.

Today, many protein extraction kits are available in the market containing the right lysis buffer, extraction buffer, and other chemicals and reagents that are required to carry out the workflow. They make the process easier and quicker with reduced hands-on time.

What is Protein Extraction Used For?

Protein extraction is one of the most common procedures performed in many life sciences labs, such as biochemistry, molecular biology, and proteomics labs. Whether it’s research or commercial production, the first step to these operations is protein purification.

Here’re a few of its applications:

  • In clinical labs, protein extraction is used to diagnose diseases, such as diabetes (by detecting the presence of insulin), or for cosmetic purposes, such as skin treatment using collagen.
  • In research, many downstream applications (especially detection methods) involve protein extraction procedures, such as biomarker discovery, gel electrophoresis, western blotting, enzyme assays, and, and immunoprecipitations.
  • Extraction of the protein of interest is crucial for determining its function, structure, and interactions in organisms.
  • Protein purification is widely used in tissue engineering and drug discovery for many purposes, such as meeting the rising demands of peptide-based biomaterials.

The detection of the protein of interest is performed through either antibody-dependent methods or spectrometry.

How Do You Extract Proteins?

A range of protein extraction protocols is available that you can choose based on the location or source of your protein. For example, when extracting proteins from tissue samples you need to homogenize the sample and there are additional steps involved. However, no such steps needed to be followed while working with cell cultures.  

The extracted protein samples are then purified using techniques like centrifugation, precipitation, filtration, and solubilization. This is followed by the refinement of the purified proteins through immunoprecipitation of affinity columns.

Via Affinity Columns

Affinity columns involve highly specific interactions between the biomolecule of interest (in this case a protein) and the resin in the column. This can include enzyme-substrate, enzyme-inhibitor, or antibody-antigen interactions, among others.. Then, the proteins are obtained by washing them and eluting them using suitable reagents.

Figure: Affinity tag protein purification.

Via Centrifugation

Centrifugation is vital to extracting proteins. During sample preparation, after the cells are lysed, the large debris or unwanted materials are removed using the technique multiple times involving some chemicals, such as denaturant. The most commonly used centrifugation technique is differential centrifugation.

Broken cellular debris and components are removed from different layers, such as cell walls and nuclear membrane (surrounding nucleus), by using centrifugation. The technique is also effective in the precipitation and extraction of nucleic acids or proteins in supernatants.

The proteins in the sample need to be broken into smaller sizes for some downstream applications, such as SDS-PAGE. High salt concentration or high/low pH buffer is used to disrupt the hydrophilic and hydrophobic interactions of proteins. The extracted protein is then precipitated from their solution and collected for further applications in labs.

Via Antibodies

ELISA, a sensitive immunoassay method, employs antibodies to detect trace amounts of antigens in bodily fluids. The enzyme-amplified reaction allows for both the identification and quantification of target proteins, eliminating the need for purification.

Additionally, by using immunohistochemistry or immunofluorescent labeling, it is possible to precisely locate and quantify the number of proteins within tissues and cells, given there are enough antibodies available.

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Protein extraction is the isolation and purification of the target protein from a variety of biological samples, such as cells, tissues, or fluids. The method is crucial for life sciences labs to obtain proteins of interest for their downstream applications.

Furthermore, the extraction of protein is also necessary for drug development and food products such as meat alternatives, protein supplements, and dairy substitutes.

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