This is of vital importance since these chemical compounds can attack and destroy the nucleic acids in your sample. For sure, compromising the integrity of your sample is the last thing on your mind, right? You can prevent this from happening by using the appropriate protease to clean up your sample.
After all the unwanted proteins and enzymes have been removed from your sample, you can then proceed with the precipitation and purification of your precious DNA sample — one that is fit for stringent DNA testing.
Image By: Alfred Hermida. Nucleic acid purification by using zirconia bead is another type of magnetic bead based purification. These microspherical paramagnetic beads have a large available binding surface and can be dispersed in solution.
This characteristic allowed thorough nucleic acid binding, washing, and elution. The total nucleic acid isolation kit, which uses this technology for the nucleic acid purification, makes use of the mechanical disruption of samples with zirconia beads in a guanidinium thiocyanate-based solution that not only releases nucleic acid but also inactivate nuclease in the sample matrix [ 30 ].
After the lysis step, dilution of samples is done by using isopropanol. Paramagenetic beads are added to the samples for the nucleic acid binding purpose. The mixture of beads and nucleic acid are immobilized on magnets and washed to remove protein and contaminants. Removal of residual binding solution is done with a second wash solution and finally the nucleic acid is eluted in low-salt buffer [ 30 ].
Solid-phase reversible immobilization paramagenetic bead-based technology has been utilized for a PCR purification system to deliver quality DNA. It requires simple protocol without centrifugation and filtration. PCR amplicons bind to paramagenetic particles which draw them out of solution, allowing contaminants such as dNTPs, primers, and salts to be rinsed away [ 31 ].
RNA with a poly-A tail attach to the oligo dT. The magnetic beads which are specially treated minimize the nonspecific binding of other nucleic acids and ensure the purity of mRNA [ 32 ]. Anion exchange resin is one of the popular examples that utilized the anion-exchange principle [ 33 ]. It is based on the interaction between positively charged diethylaminoethyl cellulose DEAE groups on the resin's surface and negatively charged phosphates of the DNA backbone.
The anion-exchange resin consists of defined silica beads with a large pore size, a hydrophilic surface coating and has a high charge density [ 34 ]. The large surface area of resin allows dense coupling of the DEAE groups. Therefore, salt concentration and pH conditions of the buffers are one of the main factors that determine whether nucleic acid is bound or eluted out from the column.
Impurities such as protein and RNA are washed from the resin by using medium-salt buffers, while DNA remains bound until eluted with a high-salt buffer [ 34 ]. The method of utilizing anion exchange materials to isolate nucleic acid has been disclosed in an invention [ 35 ], where the commercially available strong or weak positively charged anion exchanger materials were used with selected solutions of known ionic strength for adsorption and elution.
Most of the water-soluble components such as protein can be washed through the column by employing a solution with a known ionic strength for the binding of nucleic acids to the anion exchange column materials. The ionic strength for elution is generated by using known salt concentration, which mixed with a buffer to control pH strength, ideally corresponding to the lowest ionic strength at which the nucleic acids will completely elute [ 35 ].
The first step in protein purification is cell lysis. In order to purify and analyze protein efficiently, they must be first released from their host cell in a soluble form.
The plasma membrane of mammalian cells, composed of phospholipids and proteins, is easy to be disrupted [ 36 ]. In comparison, protein extraction from fungi and bacteria appears more challenging due to their stable cell wall that is stronger than the plasma membrane. Plant tissues contain a wide range of proteins which vary in their properties. Some specific factors must be taken into account when developing protein extraction protocol for plant [ 37 ]. For example, the presence of rigid cellulose cell wall must be sheared in order to release the cell contents.
Specific contaminating compounds such as phenolics and a range of proteinases may result in protein degradation or modification. Therefore, specific conditions are required for protein extraction and purification from plant [ 38 ]. Mechanical disruption techniques, such as French Press or glass beads are used to remove the cell wall, followed by detergent based extraction of total protein [ 39 ].
Ion exchange chromatography separates proteins based on their surface ionic charge using resin that are modified with either positively-charged or negatively-charged chemical groups [ 4 , 7 ].
Most proteins have an overall negative or positive charge depending on their isoelectric point pI at a given pH, which makes them possible to interact with an opposite charged chromatographic matrix [ 7 ]. If the net charge of the protein is positive at a pH below pI value, the protein will bind to a cation exchanger; at a pH above the pI value the net charge of the protein is negative and the protein will bind to an anion exchanger [ 38 ].
Proteins that interact weakly with the resins, for example a weak positively charged protein passed over resin modified with a negatively charged group, are eluted out in a low-salt buffer. On the other hand, proteins that interact strongly required more salt to be eluted.
Proteins with very similar charge characteristics can be separated into different fractions as they are eluted from the column by increasing the concentration of salt in elution buffer [ 7 ].
Ion exchange column is one of the technologies that utilized the principle of ion exchange chromatography [ 33 ]. It uses membrane-absorbent technology as a chromatographic matrix to separate proteins. The membrane absorbents in columns are stabilized cellulose-based with highly porous structure that provides proteins access to the charged surface easily. Interactions among molecules and active sites on the membrane happened in convective through-pores.
Therefore, the adsorptive membranes have the potential to maintain high efficiencies when purifying large biomolecules with low diffusion [ 33 ]. Gel filtration chromatography, also called size-exclusion or gel-permeation chromatography, separates proteins according to molecular sizes and shape and the molecules do not bind to the chromatography medium [ 39 ].
It is a process in which large molecules passes through the column faster than small molecules. Small molecules can enter all of the tiny holes of the matrix and access more of the column.
Small-sized proteins will pass through those holes and take more time to run out of the column compared with large-sized proteins that cannot get into those holes but run out directly of the column through void space in the column [ 4 , 7 ].
Gel filtration chromatography kit applies the principle of gel filtration chromatography [ 40 ]. The target sample is applied on top of the column which contained porous beads, an example of matrix in the column. The molecules get separated when the molecules pass through the column of porous beads. The separation of molecules can be divided into three main types: total exclusion, selective permeation, and total permeation limit.
Total exclusion is the part that large molecules cannot enter the pores and elute fast. For selective permeation region, intermediate molecules may enter the pores and may have an average residence time in the particles depending on their size and shape. As for total permeation limit, small molecules have the longest residence time once they enter the pores on the column [ 40 ]. An advantage of gel filtration-based chromatography is that it is suited for biomolecules that may be sensitive to pH changes, concentration of metal ions, and harsh environmental conditions [ 39 ].
Affinity chromatography depends on a specific interaction between the protein and the solid phase to affect separation from contaminants. It consists of the same steps as ion exchange chromatography [ 38 ].
It enables the purification of a protein on the basis of its biological function or individual chemical structure [ 41 ]. Proteins that have a high affinity towards the specific chemical groups such as ligands will covalently attach and bind to the column matrix while other proteins pass through the column [ 38 ].
Electrostatic or hydrophobic interactions, van der Waals' forces and hydrogen bonding are the biological interactions between ligands and the target proteins [ 41 ].
The bound proteins will be eluted out from the column by a solution containing high concentration of soluble form of the ligand [ 36 ]. A biospecific ligand that can attach to a chromatography matrix covalently is one of the requirements for successful affinity purification.
The binding between the ligand and target protein molecules must be reversible to allow the proteins to be removed in an active form [ 41 ]. After washing away the contaminants, the coupled ligand must retain its specific binding affinity for the target proteins. Some examples of biological interactions that are usually used in affinity chromatography are listed in Table 1 see [ 41 ]. Typical biological interactions used in affinity chromatography [ 42 ]. Chromatographic separation by differential affinity to ligands immobilized on a beaded porous resin is fundamental to protein research [ 42 ].
A complete kit that contains pack beaded affinity resin columns based on principle of affinity chromatography has been introduced to the market [ 42 ]. An affinity resin can be used in batch or microcentrifuge spin column format depending on the scale and type of experiment to be carried out. Furthermore, it can be packed into some sort of larger gravity-flow column as well [ 42 ].
Gel electrophoresis is a method to separate protein according to their size and charge properties. The partially purified protein from the chromatography separations can be further purified with nondenaturing polyacrylamide gel electrophoresis PAGE , or native gel electrophoresis [ 4 ]. In PAGE, the proteins are driven by an applied current through a gelated matrix [ 43 ]. The movement of protein through this gel depends on the charge density charge per unit of mass of the molecules.
The molecules with high density charge migrate rapidly. The size and shape of protein are another two important factors that influence PAGE fractionation [ 43 ].
The acrylamide pore size plays a role as a molecular sieve to separate different sizes of proteins [ 4 ]. The larger the protein, the slower it migrates as it becomes more entangled in the gel [ 43 ]. Shape is also one of the factors because compact globular proteins move faster than elongated fibrous proteins of comparable molecular mass [ 43 ]. A protein treated with SDS will usually eliminate the secondary, tertiary and quarternary structure of protein [ 4 , 7 ]. Proteins unfold into a similar rod-like shape because of the electrostatic repulsion between the bound SDS molecules.
The number of SDS molecules which bind to a protein is approximately proportional to the protein's molecular mass about 1. Each protein species has an equivalent charge density and is driven through the gel with the same force [ 43 ]. In addition, PAGE can minimize the denaturation of proteins. Many proteins still retain their biological activities after running PAGE [ 7 ]. However, larger proteins are held up to a greater degree than smaller proteins because the polyacrylamide is highly cross-linked [ 43 ].
SDS-PAGE can be used to determine the molecular mass of the mixture of proteins by comparing the positions of the bands to those produced by proteins of known size [ 43 ]. SDS used in electrophoresis resolve mixture of proteins according to the length of individual polypeptide chains [ 7 ].
First, proteins are separated according to their isoelectric point in a tubular gel. After this separation, the gel is removed and placed on top of a slab of SDS-saturated polyacrylamide.
The proteins move into the slab gel and separated according to their molecular mass [ 43 ]. Two-dimensional gel electrophoresis is suitable to detect changes in proteins present in a cell under different conditions, at different stages in development or the cell cycle, or in different organisms [ 43 ].
Southwestern blotting is a method that is used to isolate, identify, and characterize DNA-binding proteins by their ability in binding to specific oligonucleotide probes [ 44 , 45 ]. Many of the DNA-binding proteins in the cell need to be isolated individually and characterized to define the gene function [ 44 ]. Three steps are involved in this method. Next, separated proteins are transferred to a nitrocellulose filter, polyvinylidene difluoride PVDF or cationic nylon membrane [ 12 ].
The filter will then be incubated with oligonucleotide probes to analyze the adsorbed proteins [ 44 , 45 ].
Generally, the extraction or purification techniques or kits available in the market can only allow the extraction of one type of nucleic acid, either DNA or RNA, or protein from a targeted organism. A variation on the single-step isolation method of Chomczynski and Sacchi , that the guanidinium thyicyanate homogenate is extracted with phenol:chloroform at reduced pH, allows the preparation of DNA, RNA and protein from tissue or cells. This method involves the lysis of cells with guanidine isothiocyanate and phenol in a single-phase solution.
A second phase forms after the addition of chloroform where DNA and proteins are extracted, leaving RNA in the aqueous supernatant. The DNA and proteins can be isolated from the organic phase by precipitation with ethanol or isopropanol and the RNA precipitated from aqueous phase with isopropanol [ 15 ]. Several all-in-one extraction kits have been introduced in the market nowadays.
For example, a column-based extraction kit that designed to purify genomic DNA, total RNA and total protein from a single biological sample simultaneously, without the usage of toxic substances such as phenol or chloroform and alcohol precipitation [ 46 ]. It is compatible with small amounts of a wide range of cultured cells and harvested tissue of animal and human origin. The targeted sample does not need to be separated into 3 parts before the purification of DNA, RNA and protein [ 46 ].
A solution-based 3-in-1 extraction kit that is available in the market is another example of non-organic solutions kit that can extract and purify DNA, RNA and protein, from different organisms in any types and sizes [ 47 ]. Its three simple steps protocol, which takes around 15 to 30 minutes, provides a fast and easy way to do the extraction of different biomolecules. To achieve precipitation DNA.
It solubalize lipids and protiens to remove them from DNA. Sodium chloride help the the precipitation of DNA. It lyses the cell membrane and nucleus to make the extraction possible. Sodium chloride help to separate DNA from other proteins.
This means the cell has to be broken and the cytoplasmic contents released. After which, the nucleus has to be broken to release the DNA. The function of the lysis buffer is to aid in the breaking of the cell membrane or cell wall in plants.
The lysis buffer contains protease enzymes and essential salts to bring about this process. During the extraction of DNA or nucleic acids in general , there is a lot of contaminating proteins present. These contaminants must be removed. Proteinase K, which is a broad spectrum serine protease, is used in many DNA extraction protocols to digest these contaminating proteins. In addition, there may be nucleases enzymes that degrade nucleic acids present.
The addition of proteinase K degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, proteinase K is stable over a wide pH range and is well suited for use in DNA extraction. Triton X is used as a lysis buffer for DNA separation. Proteinase K, which is a broad spectrum serine protease , is used in many DNA extraction protocols to digest these contaminating proteins. Since DNA is insoluble in ethanol and isopropanol, the addition of alcohol , followed by centrifugation, will cause the DNA proteins to come out of the solution.
When DNA concentration in the sample is heavy, the addition of ethanol will cause a white precipitate to form immediately. Today, proteins are formed following instructions given by DNA deoxyribonucleic acid which in turn is synthesized by specific enzymes that are proteins. DNA contains the genetic information of all living organisms.
Proteins are large molecules made up by 20 small molecules called amino acids. Ice cold ethanol helps in increasing the yield of DNA. One more reason for using ice cold ethanol can be that the enzymes DNAses, like other enzymes are temperature sensitive and therefore DNAses are not active when the temperature is too low. The use of CTAB cetyl trimethylammonium bromide , a cationic detergent, facilitates the separation of polysaccharides during purification while additives, such as polyvinylpyrrolidone, can aid in removing polyphenols.
A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells e. Lysis buffers can be used on both animal and plant tissue cells. The function of Proteases -enzyme. Protease refers to a group of enzymes whose catalytic function is to hydrolyze peptide bonds of proteins.
They are also called proteolytic enzymes or proteinases. For example, in the small intestine, proteases digest dietary proteins to allow absorption of amino acids. Proteolytic enzyme , also called protease , proteinase, or peptidase, any of a group of enzymes that break the long chainlike molecules of proteins into shorter fragments peptides and eventually into their components, amino acids. According to invitrogen proteinase K does undergo autolysis, but that some leftover fragments still have protease activity.
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