Protein Purification Technology

Protein Purification is a series of procedures for separating proteins from a complex mixture. Its goal is to obtain highly pure, stable and active protein for downstream experiments. Purification of proteins of interest is critical to determining their function, interactions and structure. Purification can separate the proteins of the mixture from the other components and separate desired proteins from unwanted proteins.

Protein purification takes advantage of the inherent similarities and differences between different proteins. The similarity between various proteins is used to remove the contamination of non-protein substances, and the difference of each protein is used to purify the target protein from other proteins. The size, shape, charge, hydrophobicity, solubility, and biological activity of each protein will vary, and these differences can be used to extract the protein from the mixture.

• Cell lysis
  Use various enzymatic methods (protease inhibitors), chemical methods (detergents), or physical methods (sonication) to disrupt cells and denature proteins.
• Clarification
  Remove all the contaminants by filtration or centrifugation.
• Protein binding
  Isolate the protein from the extract by binding the protein molecule to a matrix.
• Elution
  Use an appropriate elution buffer to wash away all the non-specific binding on the column by changing the pH of the column and neutralizing the charged functional groups of proteins.

Fig. 1 Principle of affinity-based protein purification.

• Chromatography
  Chromatography is a common technique that separates substances according to the difference in their properties. It works on the principle of a stationary phase and a mobile phase, where the stationary phase is the resin in the column, and the mobile phase is the liquid solvent.
• Isolation
  Isolation is the simplest of all protein purification strategies. Proteins can be isolated based on the difference in the concentration gradient with ultra-centrifugation. Fractionation is based on the difference in the solubility of proteins.
• Precipitation
  The purification process of salt precipitation effectively "salt outs" or culls protein from the mixture. Precipitation is carried out by introducing high osmolarity, using ammonium sulfate as the salt.
• Immunoblotting
  Immunoblotting is a widely used technique to visualize proteins and is sometimes carried out in combination with affinity chromatography. It is a rapid and sensitive assay mainly employed for the detection of proteins.

• Preparative purification: create many purified proteins for later usage. Remove bi-products, such as host cell proteins, which could pose a patient's health risk.
• Analytical purification: yield a small amount of protein for research or analytical objectives, such as protein identification, qualification, structural, post-translational modification, and function studies.

Laboratory Centrifuges

A centrifuge is any device that applies a sustained centrifugal force—that is, a force due to rotation. The widest use of centrifuges is for the concentration and purification of materials in suspension or dissolved in fluids. Suspended particles denser than the suspending liquid tend to migrate toward the periphery, while those less dense move toward the centre.

Chromatography Columns

Columns play a central role in the performance of chromatographic process. Well-packed columns with consistent performance in the whole design space of a process will deliver consistent product recovery and separation from contaminants. Proper design of columns delivers increased resolution between peaks facilitates the packing process of multiple resin types at different bed heights, and provides scalability from process development to full-scale manufacturing.

Homogenizers / Sonicators

Homogenizers, commonly referred to as "sonicators", disrupt tissues and cells through cavitation and ultrasonic waves. Basically, a homogenizer has a tip which very rapidly vibrates, causing bubbles in the surrounding solution to rapidly form and collapse. This creates shear and shock waves which tear apart cells and particles. Homogenizers / Sonicators are great for breaking apart cells and subcellular structures in suspension. They are not good for homogenizing intact tissue.

pH Meters and Osmometers

Columns play a central role in the performance of chromatographic process. Well-packed columns with consistent performance in the whole design space of a process will deliver consistent product recovery and separation from contaminants. Proper design of columns delivers increased resolution between peaks facilitates the packing process of multiple resin types at different bed heights, and provides scalability from process development to full-scale manufacturing.

STEMart provides you with a variety of protein purification technology equipment or consumables to meet your various R&D and application needs. If you have any questions or requirements for protein purification technology, please feel free to contact us.