Protein Purification Service

2020-06-19

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Protein purification

In order to conduct in-vitro research, the target protein must first be purified from other cellular components. This process usually begins with cell lysis (the purification of secreted proteins does not require cell lysis), and the cell contents are released into the solution by destroying the cell membrane to obtain a cell lysate containing the protein of interest. The membrane lipids and membrane proteins, organelles, nucleic acids, and soluble protein-containing mixtures in the cell lysate are then ultracentrifuged.

For natural proteins, a series of purification steps may be required to obtain proteins with sufficient purity for laboratory applications. In order to simplify this process, genetic engineering is usually used to add some chemical properties to the protein of interest, making the purification process easier without changing its structure and biological activity. Usually, a “tag” containing a specific amino acid sequence is connected to the N-terminus or C-terminus of the protein of interest. For example, a sequence containing multiple consecutive histidines is called a histidine tag; when a lysate containing a histidine-tagged protein is passed through an affinity chromatography column containing nickel, histidine can chelate with nickel Therefore, it is bound to the column, and other proteins in the lysate directly flow out of the column without the histidine tag, so as to achieve the purpose of separation. Proteins obtained through genetic engineering (that is, DNA recombination) are called recombinant proteins.

Protein separation and purification method
(1) Separation and purification according to different molecular sizes. Protein is a large molecular substance, and the molecular size of different proteins is different, so some simple methods can be used to separate the protein from the small molecular substance, and the protein mixture is also separated. Methods for separation according to the size of protein molecules include dialysis, ultrafiltration, centrifugation, and gel filtration.
(2) Separation and purification according to different solubility. There are many external conditions that affect protein solubility, such as the pH, ionic strength, dielectric constant, and temperature of the solution. However, under the same conditions, different proteins have different solubility due to their molecular structure. According to the characteristics of protein molecular structure, appropriate changes in external conditions can selectively control the solubility of a component in the protein mixture to achieve The purpose of separation and purification of protein. Commonly used methods are isoelectric point precipitation and pH adjustment, protein salting and salting out, organic solvent method, two-phase extraction method, reverse micellar extraction method, etc.
(3) Separation and purification according to different charges. There are two types of methods for separating proteins according to their charge, that is, their acidity and basicity. Electrophoresis is a phenomenon in which charged particles (such as protein molecules that are not in an isoelectric point) will move toward an electrode that is opposite to its electrical property under the action of an external electric field. Polyacrylamide electrophoresis is a zone electrophoresis using polyacrylamide as a medium, and is often used to separate proteins. Ion exchange chromatography is a chromatographic method that uses ion exchangers as the stationary phase and separates them according to the difference in binding force when the component ions in the mobile phase and the counter ions on the exchanger are reversibly exchanged.
(4) Separate and purify with specific affinity to ligand. Affinity chromatography is an effective purification method that uses protein molecules to uniquely recognize their ligand molecules. In recent years, affinity chromatography technology has been widely used in the separation and purification of fusion proteins, because fusion proteins have specific binding capabilities. However, in practical work, it is difficult to separate and purify proteins with a single method, and it is often necessary to integrate several methods to purify a protein. The ideal protein separation and purification method requires higher product purity and total recovery as possible, but in fact it is difficult to balance both. With the development of biological separation technology and the continuous emergence of new biological separation technology, many new methods and methods have been provided for the separation and purification of proteins.

Classification options	Details
For separating magnetic particles in 1.5 ml or 2 ml tubes	12-Tube Magnet can be used for 6xHis-tagged protein magnetic capture experiments, or used in conjunction with Ni-NTA Magnetic Agarose Beads for purification experiments. The magnetic block consists of 12 powerful NdFeB (neodymium iron boron) disks. Each disk attracts magnetic beads in 1.5 ml or 2 ml tubes in adjacent wells. After capturing the 6xHis-tagged protein, the magnetic beads are drawn to the end of the tube, and the buffer can be changed. The magnetic beads can be resuspended away from the magnetic field to achieve thorough washing.
For separating magnetic particles in 5 x 15 ml or 3 x 50 ml tubes	15 ml/50 ml Tube Magnet is a convenient separation device, suitable for separating cells in 15 ml or 50 ml tubes, magnetic capture or immunoassay with BioMag magnetic beads. The magnetic beads are attracted to one end of the tube under the action of the magnetic field. The attractive force is greater than 20 megaoersted. When the buffer is changed or poured out, the magnetic beads will stay in the attracted position. The magnetic beads can be resuspended conveniently after removing the magnetic field.
Albumin Affinity Cartridges Removal of albumin from human and mouse plasma and serum samples using liquid chromatography	Effectively removes albumin, which facilitates the analysis of low-abundance proteins Highly specific removal of high-abundance proteins using immobilized monoclonal antibodies New design filter cartridge, easy and fast to operate Albumin Affinity Cartridges are pre-loaded with immobilized monoclonal antibodies bound to resin, which can use the principle of liquid chromatography to remove albumin from human and mouse plasma and serum samples. Helps to enrich low-abundance proteins for analysis. Procedure: The diluted sample flows slowly through the filter cartridge, during which albumin binds to the monoclonal antibody immobilized on the resin. The plasma or serum protein in the effluent does not contain albumin, and this effluent can be collected for analysis. If necessary, albumin and related proteins can be eluted from the resin using a glycine buffer with a pH of 2. Application: Albumin Affinity Cartridges can quickly and specifically remove albumin from human or mouse plasma and serum samples, which is helpful for the analysis of low-abundance proteins.
Albumin/IgG Depletion Cartridge Removal of IgG and albumin in human serum and plasma using liquid chromatography	Effectively removes IgG and albumin from human plasma and serum samples Useful for analysis of low-abundance plasma or serum proteins Immobilized monoclonal antibodies remove IgG and albumin with high specificity New design filter cartridge, easy and fast to operate Albumin/IgG Depletion Cartridges are pre-loaded with immobilized monoclonal antibodies bound to the resin, which can use liquid chromatography to remove albumin and IgG from human plasma and serum samples. Helps to enrich low-abundance proteins for analysis. Operation process: The diluted sample flows slowly through the filter cartridge, during which albumin and IgG bind to the monoclonal antibody immobilized on the resin. The plasma or serum protein in the effluent does not contain albumin and IgG, and the effluent is collected for analysis. If necessary, a pH 2 glycine buffer can be used to elute albumin, IgG and related proteins from the resin in the filter cartridge. Application: Albumin/IgG Depletion Cartridges can quickly and specifically remove albumin and IgG from human plasma and serum samples, which is helpful for the analysis of low-abundance proteins.
Allprotect Tissue Reagent Quickly stabilize DNA, RNA and protein in tissues	No need for liquid nitrogen or dry ice Stable collected tissue immediately and conveniently Long-term storage of tissues for subsequent analysis Standard sample preparation process for systems biology research Allprotect Tissue Reagent can immediately stabilize DNA, RNA and protein in tissue samples at room temperature. This stabilizes DNA, RNA, and protein to ensure reliable downstream analysis. Stabilized tissues can be transported at 15–25°C for 7 days, stored at 2–8°C for up to 12 months, and stored longer at –20°C or –80°C. Operation process: The diluted sample flows slowly through the filter cartridge, during which albumin and IgG bind to the monoclonal antibody immobilized on the resin. The plasma or serum protein in the effluent does not contain albumin and IgG, and the effluent is collected for analysis. If necessary, a pH 2 glycine buffer can be used to elute albumin, IgG and related proteins from the resin in the filter cartridge. Application: Albumin/IgG Depletion Cartridges can quickly and specifically remove albumin and IgG in human plasma and serum samples, which is helpful for the analysis of low-abundance proteins.