Isolation and Purification of Proteins Essay Example

to assist in identifying and extracting protein components (Carpentier et al., 2005). This capability stems from their amphipathic nature - featuring both a polar carboxylate group within their hydrophilic heads and lengthy alkyl-chain within their hydrophobic tails (Schmid, 1996).

Micelles, spherical clusters made up of hundreds of carboxylate ions distributed in the water phase, mimic the behavior of detergents in water (Schmid, 1996). Like the lipid bilayer forming cell membranes, the hydrophilic part of a micelle faces outward while the lipophilic tails aim inside. This similar characteristic potentially allows detergents to solubilize proteins (Carpentier et al., 2005). Substances like PBS, PBS combined with 1% Triton x-100, water, ethanol, and PBS with 2% SDS facilitate protein identification and extraction because they are examples of such detergents (Carpentier et al., 2005).

The ionic detergent Sodium dodecyl sulfate (SDS) is known for its ability to disrupt peptide bonds and has been shown to be particularly effective when used with membrane proteins (Laemmli, 1970). This detergent can break down proteins into smaller pieces, which can then be further processed using polyacrylamide gel electrophoresis (PAGE), a technique that allows the separation of these fragments based on their molecular weights (Cleveland, et al. , 1977). On another note, Triton x-100 is a form of non-ionic detergent made up either polyoxyethylene or glycosidic components in its hydrophilic head. Its unique composition enables it to interfere with the interactions among proteins and lipids as well as those within lipid groups.

The presence of this feature simplifies the isolation of membrane proteins (Laemmli, 1970). When salt levels are low, comparable to physiological measurements such as plasma salt concentrations, a multitude

of proteins is soluble (Seddon et al., 2004). Conversely, an increase in concentration escalates the salt's solubility in the solution, leading to protein salting out or precipitation. This phenomenon can vary depending on the specific salt type used, necessitating the use of detergents for additional dissolution processes (Marshal, 2011).

Inyang and Iduh's studies have demonstrated that protein solubility tends to increase along with the rise in ionic strength or sodium levels. This suggests that such a feature can be attributed to the globulins present in molecules, or enhanced performance and bonding of chloride ions with protein clusters carrying positive charges (Inyang and Iduh, 1996). Phosphate buffered saline (PBS), frequently used in numerous applications, assists in preventing proteins from denaturing by maintaining a constant pH level within the solution and fostering interactions between its water molecules and proteins (Crowther, 1995).

Alterations in the pH level of a solution can influence the structure of proteins, thereby changing their ability to interact or associate with a cell membrane (Jiang, et al., 1990). Given that proteins have charges on their surfaces, they become surrounded by water when existing in fluid solutions and act as dispersed hydrated particles (Oakley et al., 2003). Conversely, dehydrated or precipitated proteins won't readily dissolve in water without appropriate hydration.

Hence, providing sufficient hydration before solubilization is critical (Oakley et al. 2003). Certain proteins can somewhat dissolve in alcohols like ethanol, with the degree of dissolution determined by the protein's nature (Oakley et al. , 2003). Proteins interact with ethanol to reveal their non-polar side chains and internalize their peptide groups, resulting in a rod-shaped configuration with alpha-helix formations. As such, proteins

featuring alpha-helices maintain stability in ethanol while the remainder exhibit instability in most polar solvents (Oakley et al. , 2003).

The proteomics realm, a term born in 1994, shares a deep-rooted history with clinical chemistry as highlighted by Hortin et al. in 2006. Its scope encompasses the recognition, measurement, and comprehensive study of both the structure and function of proteins. Diverse approaches exist for protein quantification, with the Bradford assay outlined below being one of the most prevalent techniques, as noted by Seevaratnam et al. in 2009. The said assay harnesses the effect of the Coomassie brilliant blue (CBB) G-250 (CBBG-250) dye for its process.

The dye comes in three forms: anionic, cationic, and neutral. The anionic variant has the capacity to readily establish a complex with proteins due to Van der Waals interactions and hydrophobic forces (Compton and Jones, 1985). It is identifiable by its blue color with peak absorption between 590-595 nm (Compton and Jones, 1985). The Bradford assay technique allows for the measurement of protein quantities in micrograms through the binding of CBBG-250 to the protein. This bonding results in a color transition from red to blue which consequently changes light absorption measurable via spectrophotometry from 465 nm to 595 nm (Bradford, 1979).

A heightened intensity of the blue color indicates an increased concentration of protein (Bradford, 1979). Apart from being employed in the Bradford assay, the Coomassie blue dye also serves the purpose of staining protein bands in polyacrylamide gels which have been subjected to SDS-PAGE as it enables the visualization of protein bands (Patton, 2002). However, the Coomassie dye's interaction with arginine and lysine limits its applicability

to specific proteins (Bradford, 1979).

Despite modest success, efforts have been made to enhance the dye. The Bradford assay's strengths include consistent outcomes and swift completion (the protein binding process takes just two minutes). The dye-protein complexes created during this process remain stable in the solution for up to 60 minutes. Factors like cations and carbohydrates don't have the ability to disrupt the assay (Bradford, 1979). Alkaline agents within the mix can be managed with buffers (Bradford, 1979).

High concentrations of detergents such as SDS and Triton X-100, along with commercial glassware, may affect results by reducing the dye's color intensity due to dilution and refraction (Bradford, 1979). The use of cellular membrane components could also alter outcomes since they act as a colloidal suspension (Marshal, 2011). The Bradford assay is often utilized for evaluating protein levels by firstly diluting the protein under investigation. This is attributed to the spectrophotometer's capability of identifying concentrations ranging from 2 µg/ml to 120 µg/ml (Bradford, 1976), similar to proteins rich in arginine residues and hydrophobic components.

A graph of the absorbance curve is drawn concurrently with the absorbance of a standard volume of protein with known concentration possessing arginine and hydrophobic constituents like bovine serum albumin or BSA (Bradford, 1976). A different method for analyzing proteins, coined as the Dumbroff/Ghosh method, refrains from using spectroscopy and experiences decreased interference from detergents relative to the Bradford assay (Dumbroff, 1988). This method utilizes the absorption of the protein's solid phase on Whitman filter paper which subsequently gets stained with Coomassie blue.

As disclosed by Dumbroff (1988), proteins trapped in the filter paper manifest as differently

colored spots where the color's intensity directly corresponds to the protein quantity. This mirrors agarose gel electrophoresis used for DNA analysis, where proteins can be differentiated based on their molecular weight and charges via PAGE, or polyacrylamide gel electrophoresis. The underlying logic of this process leverages the varying travel speeds of proteins with different weights and charges through a medium like the polyacrylamide gel.

Pineiro et al. (1999) noted that staining with either Coomassie blue or silver allows for the depiction of proteins as bands in a gel. A form of electrophoresis, SDS-PAGE, operates by permitting the negatively charged SDS to dominate over the inherent charge of proteins. This facilitates the segregation of molecules based solely on their mass (Horton et al., 1996). Through silver stain usage, proteins are made visible in polyacrylamide gel electrophoresis. This method involves a reduction reaction between protein and silver ions (Ag-) (Horton et al., 1996). Silver staining procedures remain unaffected by conditions like temperature, solvent quality, and development timing (Horton et al., 1996). The CBB dye technique is frequently employed for protein staining due to its superior sensitivity and reproducibility despite possible interference from other substances present and its selectiveness towards basic amino acids only (Horton et al., 1996). Typically, CBB dye reacts with basic amino acids such as lysine, arginine, and histamine; an excess presence of these residues will lead to a more vibrant color.

The Western blot method, which incorporates gel electrophoresis, is a commonly used technique for protein extraction and recognition. In both their natural and denatured forms, proteins undergo electrophoresis before being transferred to a membrane, often nitrocellulose or PDVF (Belec, et al.,

1994). Antibodies are then introduced for detection; these antibodies have unique binding sites specifically designed for certain molecules (Belec, et al. 1994). This allows them to bind uniquely with the target protein, providing the distinct binding property necessary for isolating or identifying a specific protein within the nitrocellulose membrane (Belec, et al., 1994).

The Western blotting technique is a method utilized for identifying particular proteins in a specific sample. This detection uses antibodies that are produced by the immune system. These antibodies find and attach to epitopes, which are unique areas on protein surfaces (Aroor et al., 2010). Epitopes, also known as antigenic determinants, can be either continuous or discontinuous. Continuous epitopes typically consist of three to four neighboring amino acids within a small part of the primary sequence. In contrast, discontinuous epitopes comprise residues that may not be adjacent in the main sequence but merge together in the protein's folded native structure (Aroor et al., 2010). The transfer phase from an SDS-PAGE gel onto a Nitrocellulose/PVDF filter during this method ensures maintenance of protein configuration. This association between the protein and nitrocellulose/PVDF filter is referred to as non-covalent binding. Methanol is used to prime the PVDF membrane for this process (Aroor et al., 2010).

The protein-transferred filter undergoes exposure to a primary antibody, which is utilized to pinpoint the position of the target protein. The use of a secondary antibody serves to demonstrate the binding of the primary antibody to its antigen. The protein products can be visualized due to a secondary antibody that is distinctively labeled for the primary antibody, including anti-Immunoglobulin G (anti-JgG), for instance. The secondary antibody may be

subject to radioactive labeling with exposure to the western blotting technique and film, or it could be labeled with a fluorescent compound that is identifiable and quantifiable (Aroor et al. 2010).

Mass spectroscopy (MS) is a technique that simplifies the process of protein identification. It operates on the principle of determining the mass-to-charge ratio of charged particles (Moyers and McDonald 2006). In practice, this involves disintegrating a sample of peptide molecules into ions using a beam of electrons. The resulting ions are then categorized according to their mass-to-charge ratio and detected through MS (Irene, et al., 2011). Essentially, it includes stages such as preparing the sample, performing chromatographic processing, ionizing the sample, and interpreting data.

Advancements in proteomics have been significantly influenced by enhancements in Mass Spectrometry (MS) as indicated by Moyers and McDonald 2006. The research aimed to achieve two goals. Firstly, it investigated the impact of various solvents such as ethanol, water, PBS, PBS+1% Triton x-100, and PBS+2% SDS on protein solubilization. Proteins dissolved in these mediums were quantified using Bradford and Ghosh/Dumbroff techniques. Subsequently, SDS PAGE electrophoresis was employed to differentiate polypeptides available within the mixture which were later visualized using coomassie brilliant blue and silver staining methods.