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Posted on Thu, Feb 20, 2020

Protein Extraction Protocol

Proteomics is the study of the protein in an organism. Protein is a fundamental building block of life, and proteins are the workhorses within and between cells. Biochemical pathways are built out of enzymes and ligands—without them nothing would be accomplished; plants wouldn’t produce glucose, animals wouldn’t be able to digest food, the immune system would cease to exist, and all other biological processes would grind to a halt. The fundamental importance of proteins for life makes them an important topic of study. The first step in understanding protein structure and function is to extract them. Protein extraction is the process of isolating and purifying protein from samples of whole tissue, cell cultures, or biological fluids. The protein extraction protocol used is tailored to match the starting material and the end goals of the assay. Considering the goal of the experiment is extremely important when developing a protein extraction protocol because certain buffer choices (such as high salt, high detergent formulations) can ruin an experiment when higher order protein structure and function needs to be preserved.

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Protein structure

In their most fundamental state, proteins are long strings of amino acids forming the primary protein structure. The order of amino acids and the length of these chains is dictated by the genetic code. Secondary protein structure is governed by hydrogen bonding and yields secondary structure in the form of alpha helices and beta sheets. From here the protein adopts a tertiary structure that is directed by amino acid side chain interactions. Quaternary structure appears when individual proteins complex together to form a larger protein consisting of more than one amino acid chain. It is important to understand that proteins get their shape from chemical interactions between the amino acids comprising them, and that protein function is determined by protein shape. Denaturation is the process of a protein losing its shape due to disruption of the chemical bonds building its secondary, tertiary, and quaternary structure. Protein denaturation leads to loss of protein-protein interactions and function, and must be avoided at all costs when designing a protein extraction protocol for isolation of proteins in functional binding assays such as ELISA or co-immunoprecipitation. The best way to avoid this is to use gentle buffers without concentrated detergents, high salt content, or extreme pH (highly basic or acidic). 

Protein Extraction Protocol

The process of protein extraction starts with very crude samples that are cleaned up through filtration, centrifugation, solubilization and precipitation, and refined with techniques such as affinity columns and immunoprecipitation. A general workflow proceeds as follows:

  1. Lyse cells by sonication or homogenize tissue by freezing and grinding or homogenizing. This process breaks cell walls and frees proteins into the sample. Tissue samples can then be filtered to remove large debris. 
  2. Centrifuge sample to remove cellular debris. This is usually performed in an ultracentrifuge via differential centrifugation or density gradient centrifugation. This step can be particularly useful for extracting specific compartmental proteins such as exosomal proteins
  3. For protein that will be used for a denatured gel electrophoresis or a western blot—use acetone to precipitate proteins.
  4. Resuspend sample in an appropriate protein extraction buffer. This buffer will contain an appropriate amount of salt, will contain a buffering agent to maintain pH 6-8, and will contain any necessary detergents, reducing agents, denaturants, or protease inhibitors. Detergents help to solubilize poorly soluble proteins such as integral membrane proteins that are used to a hydrophobic environment and to prevent non specific interactions. Reducing agents decrease oxidative damage. Denaturants are used only when the goal is to denature proteins for analysis of molecular weight such as SDS Page. Protease inhibitors prevent degradation by proteases once the membranes are damaged and everything is suddenly all mixed up together. 
  5. Biomagnetic separation may be used as an optional step for further refinement of target protein solution. This technique is able to specifically enrich target protein due to a “lock-and-key” match between the target protein and the capture protein conjugated to magnetic nanoparticles. Once the capture protein and the target protein are bound together they are retained by a magnetic separation rack while the buffer is exchanged. The result is a highly enriched population of extracted target protein. 
  6. Measure protein concentration via absorbance at 280 nm, Lowry Assay, Bradford Assay, or Bicinchoninic Assay (BCA)
  7. Store protein extract at -80ºC or -20ºC until needed for further analysis.

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