Protein purification is a process aimed at separating one or several proteins from a complex mixture. This technique is widely used in biochemical research applications. Purification of the protein of interest is fundamental to the study of protein function, structure, and interactions. We offer a basic protocol for protein purification that focuses on four basic steps, including cell lysis, protein binding to the matrix, washing, and elution. Our goal is to keep as much functional protein as possible while removing as many contaminants as possible.
|Stages||Solutions and Reagents|
|Sample Preparation||Cell lysis buffer and washing buffer|
|Purification||Affinity chromatography matrices, ligands, equilibration buffer, washing buffer, elution buffer|
There are several common methods of protein purification, including salinization, dialysis, ion exchange, gel filtration, and affinity chromatography. Affinity chromatography is one of the most reliable and commonly used methods. This takes advantage of the specific and reversible binding of proteins to matrix-binding ligands.
Figure 1: Live Cell Imaging Procedure
Perform crude protein sample preparation, including cell harvesting, cell crushing, and clarification. First, isolate and harvest cells from the culture medium. Then perform cell fragmentation, usually using enzymatic, detergent, and sonication methods. Finally, the separation of proteins is achieved by filtration or centrifugation.
Next, the proteins in the crude extract can be initially purified by removing impurity proteins through salinization, dialysis, and gel filtration steps. You can choose a purification protocol with different principles depending on your sample.
Select porous carrier materials such as agarose, polyacrylamide, cellulose, and silica to make affinity chromatography matrices. Select affinity chromatography ligands according to the protein molecules to be separated. The ligands are covalently immobilized or adsorbed on the surface of the affinity chromatography matrix by biological interactions. Fill the column with the matrix to form the stationary phase. Apply the sample to the column. As the protein mixture passes through the chromatographic column, proteins with immobilized substrate binding sites bind to the immobilization.
Wash the column with wash buffer immediately after the sample enters the column. Impurity protein molecules that have not been bound are washed away. Then elute the non-specifically bound proteins with an elution buffer. Then, use another elution buffer to elute the specifically bound proteins. After all the specifically bound proteins are eluted from the column, wash the column with eluent.
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No protein in the eluate
Protein does not bind
We are committed to the success of your protein purification. We hope the information above will put you on the right path in your protein research. If you still have any questions, please contact our technical support staff for additional online resources.
For research use only. Not intended for any clinical use.