Oct
18
What would be a popular and useful technique that can be used in biochemistry laboratory?
Filed Under Biochemistry
I have to make a presentation about a technique that is used in biochemistry lab. such as PCR, Elisa…. (those are already taken)
Anyone who can tell me a cool, useful topic which would gimme a good point in class?
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Safety in the lab.
Protein purification
Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is vital for the characterisation of the function, structure and interactions of the protein of interest. The starting material is usually a biological tissue or a microbial culture. The various steps in the purification process may free the protein from a matrix that confines it, separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps exploit differences in protein size, physico-chemical properties and binding affinity.
Purpose
Purification may be preparative or analytical. Preparative purifications aim to produce a relatively large quantity of purified proteins for subsequent use. Examples include the preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g. soy protein isolate), and certain biopharmaceuticals (e.g. insulin). Analytical purification produces a relatively small amount of protein for a variety of research or analytical purposes, including identification, quantification, and studies of the protein’s structure, post-translational modifications and function. Among the first purified proteins were urease and Concanavalin A.
Strategies
Recombinant bacteria can be grown in a flask containing growth media.Choice of a starting material is key to the design of a purification process. In a plant or animal, a particular protein usually isn’t distributed homogeneously throughout the body; different organs or tissues have higher or lower concentrations of the protein. Use of only the tissues or organs with the highest concentration decreases the volumes needed to produce a given amount of purified protein. If the protein is present in low abundance, or if it has a high value, scientists may use recombinant DNA technology to develop cells that will produce large quantities of the desired protein (this is known as an expression system). Recombinant expression allows the protein to be tagged, e.g. by a His-tag, to facilitate purification, which means that the purification can be done in fewer steps. In addition, recombinant expression usually starts with a higher fraction of the desired protein than is present in a natural source.
An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isolectric points by running them through a pH graded gel or an ion exchange column. Second, proteins can be separated according to their size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified by using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein identity. This is very useful for scientific purposes and the detection limits for protein are nowadays very low and nanogram amounts of protein are sufficient for their analysis.
Evaluating purification yield
The most general method to monitor the purification process is by running a SDS-PAGE of the different steps. This method only gives a rough measure of the amounts of different proteins in the mixture, and it is not able to distinguish between proteins with similar molecular weight.
If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property can be used to detect and quantify the specific protein, and thus to select the fractions of the separation, that contains the protein. If antibodies against the protein are available then western blotting and ELISA can specifically detect and quantify the amount of desired protein. Some proteins function as receptors and can be detected during purification steps by a ligand binding assay, often using a radioactive ligand.
In order to evaluate the process of multistep purification, the amount of the specific protein have to be compared to the amount of total protein. The latter can be determined by the Bradford total protein assay or by absorbance of light at 280 nm, however some reagents used during the purification process may interfere with the quantification. For example, imidazole (commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA) assay for total protein quantification. Impurities in low-grade imidazole will also absorb at 280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance.
tagged protein
Adding a tag to the protein such as RuBPS gives the protein a binding affinity it would not otherwise have. Usually the recombinant protein is the only protein in the mixture with this affinity, which aids in separation. The most common tag is the Histidine-tag (His-tag), that has affinity towards nickel or cobalt ions. Thus by immobilizing nickel or cobalt ions on a resin, an affinity support that specifically binds to histidine-tagged proteins can be created. Since the protein is the only component with a His-tag, all other proteins will pass through the column, and leave the His-tagged protein bound to the resin. The protein is released from the column in a process called elution, which in this case involves adding imidazole, to compete with the His-tags for nickel binding, as it has a ring structure similar to histidine. The protein of interest is now the major protein component in the eluted mixture, and can easily be separated from any minor unwanted contaminants by a second step of purification, such as size exclusion chromatography or RP-HPLC.
Another way to tag proteins is to add an antigen peptide to the protein, and then purify the protein on a column containing immobilized antibody. This generates a very specific interaction usually only binding the desired protein.
When the tags are not needed anymore, they can be cleaved off by a protease. This often involves engineering a protease cleavage site between the tag and the protein.
Methods of protein purification
The methods used in protein purification, can roughly be divided into analytical and preparative methods. The distinction is not exact, but the deciding factor is the amount of protein, that can practically be purified with that method. Analytical methods aim to detect and identify a protein in a mixture, where as preparative methods aim to produce large quantities of the protein for other purposes, such as structural biology or industrial use. In general, the preparative methods can be used in analytical applications, but not the other way around.
Extraction
Depending on the source, the protein has to be brought into solution by breaking the tissue or cells containing it. There are several methods to achieve this: Repeated freezing and thawing, sonication, homogenization by high pressure or permeabilization by organic solvents. The method of choice depends on how fragile the protein is and how sturdy the cells are. After this extraction process soluble proteins will be in the solvent, and can be separated from cell membranes, DNA etc. by centrifugation. The extraction process also extracts proteases, which will start digesting the proteins in the solution. If the protein is sensitive to proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow down proteolysis.
Precipitation and differential solubilization
In bulk protein purification, a common first step to isolate proteins is precipitation with ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of ammonium sulfate and collecting the different fractions of precipitate protein. One advantage of this method is that it can be performed inexpensively with very large volumes.
The first proteins to be purified are water-soluble proteins. Purification of integral membrane proteins requires disruption of the cell membrane in order to isolate any one particular protein from others that are in the same membrane compartment. Sometimes a particular membrane fraction can be isolated first, such as isolating mitochondria from cells before purifying a protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate (SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein’s native conformation during complete purification.
Ultracentrifugation
Main article: Ultracentrifuge
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle) containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated at high speeds, the angular momentum yields an outward force to each particle that is proportional to its mass. The tendency of a given particle to move through the liquid because of this force is offset by the resistance the liquid exerts on the particle. The net effect of “spinning” the sample in a centrifuge is that massive, small, and dense particles move outward faster than less massive particles or particles with more “drag” in the liquid. When suspensions of particles are “spun” in a centrifuge, a “pellet” may form at the bottom of the vessel that is enriched for the most massive particles with low drag in the liquid. The remaining, non-compacted particles still remaining mostly in the liquid are called the “supernatant” and can be removed from the vessel to separate the supernatant from the pellet. The rate o
siRNA , knockout mice , and microarrays seem to be all the rage nowadays.
Or go with basic techniques like western blotting, flow cytometry or fluorescent microscopy, which are skills useful in almost any lab.