Protein analysis and purification

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Spinning a sample twice as long doesn't mean the particle of interest will go twice as far, in fact, it will go significantly further. However, when the proteins are moving through a sucrose gradient, they encounter liquid of increasing density and viscosity. A properly designed sucrose gradient will counteract the increasing centrifugal force so the particles move in close proportion to the time they have been in the centrifugal field.

Samples separated by these gradients are referred to as "rate zonal" centrifugations. 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. An analytical purification generally utilizes three properties to separate proteins. First, proteins may be purified according to their isoelectric 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. Usually a protein purification protocol contains one or more chromatographic steps. The basic procedure in chromatography is to flow the solution containing the protein through a column packed with various materials.

Different proteins interact differently with the column material, and can thus be separated by the time required to pass the column, or the conditions required to elute the protein from the column.

Many different chromatographic methods exist:. Chromatography can be used to separate protein in solution or denaturing conditions by using porous gels. This technique is known as size exclusion chromatography. The principle is that smaller molecules have to traverse a larger volume in a porous matrix.

Tools and Resources for Protein Expression, Extraction and Isolation

Consequentially, proteins of a certain range in size will require a variable volume of eluent solvent before being collected at the other end of the column of gel. In the context of protein purification, the eluent is usually pooled in different test tubes. All test tubes containing no measurable trace of the protein to purify are discarded. The remaining solution is thus made of the protein to purify and any other similarly-sized proteins. HIC media is amphiphilic, with both hydrophobic and hydrophilic regions, allowing for separation of proteins based on their surface hydrophobicity.

Target proteins and their product aggregate species tend to have different hydrophobic properties and removing them via HIC further purifies the protein of interest. In pure water, the interactions between the resin and the hydrophobic regions of protein would be very weak, but this interaction is enhanced by applying a protein sample to HIC resin in high ionic strength buffer. The ionic strength of the buffer is then reduced to elute proteins in order of decreasing hydrophobicity.

Ion exchange chromatography separates compounds according to the nature and degree of their ionic charge. The column to be used is selected according to its type and strength of charge. Anion exchange resins have a positive charge and are used to retain and separate negatively charged compounds anions , while cation exchange resins have a negative charge and are used to separate positively charged molecules cations.

Before the separation begins a buffer is pumped through the column to equilibrate the opposing charged ions. Upon injection of the sample, solute molecules will exchange with the buffer ions as each competes for the binding sites on the resin. The length of retention for each solute depends upon the strength of its charge. The most weakly charged compounds will elute first, followed by those with successively stronger charges. Because of the nature of the separating mechanism, pH, buffer type, buffer concentration, and temperature all play important roles in controlling the separation. Ion exchange chromatography is a very powerful tool for use in protein purification and is frequently used in both analytical and preparative separations.

Free-flow electrophoresis FFE is a carrier-free electrophoresis technique that allows preparative protein separation in a laminar buffer stream by using an orthogonal electric field. Affinity Chromatography is a separation technique based upon molecular conformation, which frequently utilizes application specific resins.

These resins have ligands attached to their surfaces which are specific for the compounds to be separated.

Protein Analysis Purification Benchtop by Ian Rosenberg, Hardcover - AbeBooks

Most frequently, these ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and key" fit between the ligand and its target compound makes it highly specific, frequently generating a single peak, while all else in the sample is unretained. Many membrane proteins are glycoproteins and can be purified by lectin affinity chromatography.

Detergent-solubilized proteins can be allowed to bind to a chromatography resin that has been modified to have a covalently attached lectin. Proteins that do not bind to the lectin are washed away and then specifically bound glycoproteins can be eluted by adding a high concentration of a sugar that competes with the bound glycoproteins at the lectin binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that is hard to compete with sugars, and bound glycoproteins need to be released by denaturing the lectin. A common technique involves engineering a sequence of 6 to 8 histidines into the N- or C-terminal of the protein.

The polyhistidine binds strongly to divalent metal ions such as nickel and cobalt.

The protein can be passed through a column containing immobilized nickel ions, which binds the polyhistidine tag. All untagged proteins pass through the column. The protein can be eluted with imidazole , which competes with the polyhistidine tag for binding to the column, or by a decrease in pH typically to 4.

An Overview of Protein Purification Methods

While this procedure is generally used for the purification of recombinant proteins with an engineered affinity tag such as a 6xHis tag or Clontech's HAT tag , it can also be used for natural proteins with an inherent affinity for divalent cations. Immunoaffinity chromatography uses the specific binding of an antibody -antigen to selectively purify the target protein. The procedure involves immobilizing a protein to a solid substrate e.

The target protein can be eluted by changing the pH or the salinity. The immobilized ligand can be an antibody such as Immunoglobulin G or it can be a protein such as Protein A. Because this method does not involve engineering in a tag, it can be used for proteins from natural sources. Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then purify the protein on a column or by incubating with a loose resin that is coated with an immobilized antibody. This particular procedure is known as immunoprecipitation.

Immunoprecipitation is quite capable of generating an extremely specific interaction which usually results in binding only the desired protein. The purified tagged proteins can then easily be separated from the other proteins in solution and later eluted back into clean solution. 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.

High performance liquid chromatography or high pressure liquid chromatography is a form of chromatography applying high pressure to drive the solutes through the column faster. This means that the diffusion is limited and the resolution is improved.

Protein Purification and Analysis

The most common form is "reversed phase" HPLC, where the column material is hydrophobic. The proteins are eluted by a gradient of increasing amounts of an organic solvent , such as acetonitrile. The proteins elute according to their hydrophobicity. After purification by HPLC the protein is in a solution that only contains volatile compounds, and can easily be lyophilized.

Protein Purification Product Groups

At the end of a protein purification, the protein often has to be concentrated. Different methods exist.

If the solution doesn't contain any other soluble component than the protein in question the protein can be lyophilized dried. This is commonly done after an HPLC run. This simply removes all volatile components, leaving the proteins behind. Ultrafiltration concentrates a protein solution using selective permeable membranes.

The function of the membrane is to let the water and small molecules pass through while retaining the protein. The solution is forced against the membrane by mechanical pump, gas pressure, or centrifugation. 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 apparent 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 has 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 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.

SPR can detect binding of label free molecules on the surface of a chip. Protein Analysis and Purification Benchtop Techniques. Front Matter Pages i-xxi.

Meetings & Courses Program

An Overview of this Manual. Pages Protein Structure. Tracking the Target Protein. Electrophoretic Techniques. Getting Started with Protein Purification. Membrane and Particulate-Associated Proteins. Transfer and Detection of Proteins on Membrane Supports. Peptide Mapping and Microsequencing. Modified Proteins and Peptides. Recombinant Protein Techniques. Back Matter Pages


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