Proteins are macromolecules which are made up of amino acids. Proteins perform various functions like enzyme catalytic, haemoglobin transport oxygen etc. It is an important part of our diet and is the basic building blocks of cells. Proteins are extracted from fish samples utilizing a method known as acetonitrile extraction technique.
What is protein extraction?
Protein extraction is the process of isolating and separating the protein from cells. It usually involves breaking up the cells, inactivating degrading enzymes, removing nucleic acids and other cell components, and purifying the protein for further study.
Protein extraction methods are designed to isolate a specific group of proteins or all proteins from a cell, tissue or organism. This step is essential for downstream applications such as protein quantification, crystallization, proteomics studies and protein sequence analysis.
In protein extraction protocols, many factors must be taken into consideration:
Cell type
Protein extraction methods vary depending on whether the target is prokaryotic or eukaryotic cells. For example, it is important to inactivate RNase activity when extracting RNA from eukaryotic cells because RNA degradation may occur during sample preparation.
Cell types also differ in their resistance to lysis by chemical reagents or mechanical disruption techniques. The pH of the buffer solution used can determine whether proteins remain soluble in the extract or precipitate out of solution.
Sample source
The sample source affects not only the type of cell being extracted but also the type of buffer used in each step of the procedure. For example, a nuclear lysis buffer should be used when preparing an extract that consists of nuclear proteins
Purpose of protein extraction
Protein extraction is the process of extracting protein from a sample. The extraction is done to isolate and purify proteins from the cell lysate. The goal of protein extraction is to break the non-covalent bonds of the protein, so that the protein can be used for various applications.
Protein samples are extracted from tissues, cells, or biological fluids. The amount of protein needed for each application varies. Proteins can be extracted from different organisms such as bacteria, plants, insects, and animals etc.
The main purpose of protein extraction is to separate the proteins from other components in order to study their function or structure. In some cases, organisms are grown in a controlled environment and then treated with chemicals to break down cellular structures so that proteins can be extracted.
These proteins are then analyzed by methods such as Western blotting, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, and N-terminal sequence analysis. In addition, fluorescent techniques such as fluorescence microscopy and flow cytometry can also be used to analyze these samples.
Protein extraction buffer
The first step in protein extraction is to reduce the viscosity of the sample. For example, blood must be centrifuged to separate the cell fraction from the plasma; tissue samples must be homogenized, and certain types of cells may need to be lysed. The resulting extract contains a mixture of proteins in solution.
Protein extraction buffer:
The next important step is to destabilize the cells or tissues so that soluble proteins will disperse into solution. In most cases, this can be accomplished by adding a chaotropic agent, such as guanidine HCl, urea or detergent (e.g., sodium dodecyl sulfate).
A chelating agent such as EDTA is often added next to chelate divalent ions (e.g., Mg2+ and Ca2+), which are involved in some cellular structures and activities and can remain complexed with proteins after denaturing conditions are applied.
The protein extraction buffer must adjust the pH of the sample so that all of the proteins are in their biologically functional forms. This is typically achieved using Tris-HCl or phosphate buffer at pH 7–8.
Finally, reducing agents, such as dithiothreitol (DTT) or β
Protein extraction methods
Protein extraction methods for fish samples
The most common method for protein extraction is homogenization of the tissue in solubilizing buffers. The most commonly used solubilizing buffer is Tris-HCl, with a pH of approximately 8.0 and a concentration of 1–2 M. This buffer may be supplemented with other reagents, such as urea, sodium dodecyl sulfate (SDS), bromophenol blue, and β-mercaptoethanol (β-ME).
The addition of SDS results in denaturation and unfolding of the proteins, which can help to promote protein solubilization. Urea also helps to denature and unfold the proteins but tends to lead to more protein aggregation than SDS.
To aid in the denaturation process, guanidine hydrochloride can be added; this is particularly useful for membrane proteins. The addition of dithiothreitol (DTT) or β-ME helps to prevent oxidation and degradation of the proteins during the extraction process. Additionally, protease inhibitors may be included with the extraction buffer to prevent proteolysis during the extraction.
Protein extraction in Tissue samples
Protein extraction in tissue samples is a technique used to extract proteins from animal and plant tissues. The procedure is necessary to prepare samples for several downstream applications such as immunoblotting, ELISA, and enzyme activity assays. Protein extraction may use different techniques depending on the type of protein being extracted.
Proteins are extracted from various sources like plants and animals. The first step in protein extraction is homogenization of the tissue using a buffer solution. The homogenized sample is then centrifuged to remove cellular debris, leaving behind the soluble protein fraction in the supernatant.
One of the biggest challenges in extracting proteins from tissue samples is excess lipids, carbohydrates and nucleic acids that can interfere with downstream applications, or can cause difficulty in further purification steps. These contaminants can be removed by several techniques including precipitation, dialysis and chromatography steps like salting out or ion-exchange chromatography.
Last Words
The fish waste contains the protein down to 10% of it, but why not extracting all the protein from the fish. In order to extracting the protein from fish, you have to activate it first by romping them with each other, and then using microbes to consume those proteins from the fish.