Mammalian Active Protein Extraction Reagent

General Protocol

Experiment Preparation

Take an appropriate amount of Mammalian Active Protein Extraction Reagent and add protease and/or phosphatase inhibitors as needed. Mix and keep on ice.

1. Adherent cells

1) Remove culture medium. If phenol red or other medium components may interfere with downstream analysis, wash cells 1-2 times with PBS.

2) Add an appropriate volume of extraction reagent, mix gently, and incubate on ice for 2-10 min.

3) Collect the lysate and centrifuge at 14,000 × g for 5-10 min at 4°C.

4) Transfer the supernatant to a new tube for downstream analysis or store at -80°C.

Recommended volumes for different culture vessels:

2. Suspension cells

1) Collect cells by centrifugation at 2,500 × g for 10 min at 4°C and gently disperse the pellet.

2) Add an appropriate volume of extraction reagent and incubate on ice for 2-10 min.

3) Centrifuge at 14,000 × g for 5-10 min at 4°C and collect the supernatant.

3. Tissue samples

1) Cut tissue into small pieces or freeze in liquid nitrogen and grind into powder.

2) Add 200-400 μL extraction reagent per 20 mg tissue, homogenize thoroughly, and incubate on ice for 2-10 min.

3) Centrifuge at 14,000 × g for 5-10 min at 4°C and collect the supernatant.

Note: Perform all steps on ice or at 4°C whenever possible. If the extracted active protein will be used for His-tag purification, avoid EDTA-containing inhibitors. If the lysate is highly viscous, nuclease treatment or a larger extraction volume may be used.