How to get pure protein from tissue?

2017-08-28 21:54:18 GMT+0800

As the body and the undertaker of life activity, protein plays an important role in life.

Proteins can be divided into structural proteins and functional proteins, as the name suggests, and structural proteins are the basic structures that make up organisms, such as cell skeletons and membrane proteins.

Functional proteins are essential for life activities;

For example, enzymes, transcription factors, antibodies, some hormones, etc.

After decades of development, the protein research approach has been taken from a single protein function to the dimension of proteomics.

Since I am a plant research object, I have been using arabidopsis as a model to talk about the operation of proteins.

So we're going to start with a simple way of looking at proteins, so you have to get a pure protein from the tissue. How do you do that?

1.Principle

Total protein or non-denatured tissue total protein of the group:

A. Extraction of total denatured proteins

Inside the cells of other impurities removed, only keep protein at the same time, you can add degeneration in extract of surfactants (such as: SDS sodium dodecyl sulfate) is used to break the cell + physical processing (such as liquid nitrogen grinding), the release of intracellular proteins, again through the centrifugal free protein and other impurities and cell debris separation, from the qing dynasty is the cell total protein.

B. The extraction of total protein from non-transgendered tissues

Inside the cells of other impurities removed, only keep protein at the same time, you can add the degeneration of surfactant in extract (such as: Trion - X100 qu tong X100) is used to break the cell + physical processing (such as liquid nitrogen grinding), the release of intracellular proteins, you also need to add some protease inhibitors, to prevent protein by enzyme degradation in the operation.

The free protein is separated from other impurities and cell fragments by centrifugation, and the total protein of cells is absorbed. (note: all of the proteins must be operated at 4 degrees in order to prevent the proteinosis).

2. Extraction procedure

Here is a method to extract the total protein extraction of plant.

1. Take the arabidopsis material in the mortar, and add 200 mu l Media A and 40 mu l 25% Triton - X100 solution to fully grind. This process is carried out in liquid nitrogen or ice;

2.4 ℃ 12000 RPM centrifuge for 10 min, absorbing supernatant;

The concentration of the extracted protein was measured by using the protein, and the OD600 was used to quantitatively.

The attached:

MediaA solution formula: (500ml)

1 m Tris HCl (pH8.6) 12.5 mL