If so, how can we confirm whether the protein is purified and how to increase its purity?
Proteins
We generally tend to think of proteins only from a dietary lens, as a component of what we eat. However, they are among the most important and abundant organic macromolecules in the human body, with diverse structures and functions. Every cell contains thousands and thousands of proteins, each with specific functions. Some help in the formation of cellular membrane or walls, some help the cell to move, others act as messages or signals and flow seamlessly from one cell to another, carrying information.
Protein Expression
The method by which living organisms synthesize proteins and further modify and regulate them is called protein expression. Protein expression plays a significant role in several types of research and is highly utilized in molecular biology, biochemistry, and protein research laboratories.
Can we purify mCherry protein (His-tag attached) with immobilised metal ions affinity chromatography (Ni-IDA)? If so, how can we confirm whether the protein is purified and how to increase its purity?
Chromatography
- In 1906, chromatography was introduced by Michael Tswett to separate chlorophyll pigments.
- It is a technique of separation of substances on the basis of their partition coefficients in two phases which are immiscible.
- One phase is called a stationary phase and another one is the mobile phase.
- Separation occurs according to the affinity of substances to mobile or stationary phase.
Metal Ions Affinity Chromatography
- It is a technique in which the molecule of interest is dissolved in the mobile phase and in the stationary phase metals are bound.
- It is also called IMAC (Immobilized Metal Ion Affinity Chromatography) in which protein of interest are separated due to the binding of certain amino acids to metals on the stationary phase. Metals are immobilized in the stationary phase.
Yes, it is possible to purify mCherry Protein which is His-tag with Metal Affinity Chromatography.
- In most cases, histidine is exploited to bind the metal attached to the column.
- Copper, cobalt, and nickel are generally immobilized on the column.
- When a histidine-tagged protein comes in contact with nickel immobilized on the column, the histidine will chelate the metal ion through coordinate bonding. Whereas the protein which is not tagged with histidine will bind weakly to the nickel, hence can be washed off easily after washing with elution buffer. On the contrary, His-tag Protein will remain bound to the column and can be separated by this affinity chromatography.
Step by step
Solved in 4 steps
