Protein Extraction

Most proteins are soluble in water, dilute salt, dilute acid or alkali solution, and a few proteins bound with lipids are soluble in ethanol, acetone, butanol and other organic solvents. Therefore, different Liquid–liquid extraction can be used to extract and purify proteins and enzymes. Generally speaking, alkaline proteins are extracted using slightly acidic extracts, while acidic proteins are extracted using slightly alkaline extracts.

• Extract proteins from cells

Cell suspensions were centrifuged at 4 ° C at 2000 xg for 5-7 min. Collect the cells at the bottom of the tube and discard the supernatant.

The cells were added to cold PBS and centrifuged at 4 ℃ at 2000 xg for 5-7 minutes for washing.

Cold lysis buffer was added to the cell particles, appropriate amount of protease inhibitor and phosphatase inhibitor was added (ready for use), ultrasonication or agitate the contents in microcentrifuge tubes for 30 min at 4 ℃.

Centrifuge at 4 ℃ at 16000 xg for 20 min. Discard the precipitation, collect the supernatant in a new tube, and place it on the ice.

• Extract proteins from tissues

Dissect tissues of interest on the ice. The tissue is transferred to a microcentrifuge tube and soaked in liquid nitrogen for rapid freezing.

For every 5 mg of tissue, 300 µL of cold lysis buffer was added, appropriate amount of protease inhibitor and phosphatase inhibitor (ready for use) were added and homogenized with electric homogenizer. 300-600 µL of lysis buffer is added during the homogenization process.

Stir at 4 ℃ for 2 hours.

Centrifuge at 4 ° C at 16000 xg for 20 min. Discard the precipitation, collect the supernatant in a new tube, and place it on the ice.

The total protein concentration of the sample was normalized.

A small amount of lysate was taken for protein estimation test.

By comparing with the standard, the protein concentration of the unknown sample is determined, ensuring that the standard is diluted into the same buffer as the unknown sample. Protein content can be estimated using Coomassian assay reagents, BCA tests, or 280 nm absorbance.

The appropriate volume of lysate is transferred into the microcentrifuge tube so that all samples contain the same total protein concentration.

Add enough freeze lysis buffer so that all lysates reach the same volume.

1. The whole process should be carried out in low temperature or on ice.

2. The RIPA lysate can extract whole proteins, and if specific proteins are to be extracted, they can be extracted by a kit specifically designed to extract such proteins.

3. For SDS denaturation electrophoresis, SDS can be added to denaturate the whole protein and protease at the time of protein extraction.