Nuclei / Plasma Protein Extraction Kit

To ensure the proper handling of reagents and the preparation of working solutions for protein extraction, certain steps need to be followed. After dissolving all the solutions in the room temperature melting kit, with the exception of the structural protein extract, it is important to place the other reagents on ice immediately.
To prepare the working solution for cytoplasmic protein extraction, you should estimate the amount of cytoplasmic protein extraction required for your experiment based on the number of treated samples. Then, add phosphatase inhibitor II and protease inhibitor PMSF at a ratio of 1:50 and 1:100, respectively.
Similarly, for the preparation of the nuclear protein extraction solution, estimate the amount needed for the experiment and add phosphatase inhibitor II and protease inhibitor PMSF at the same ratio.
When dealing with cultured cell samples, follow these steps:
1. For cytoplasmic protein extraction:
- Add 2.0 mL of cytoplasmic protein extraction working solution (2.0 × 10^7 cells) and shake it at 4°C for 20 minutes.
- Prepare a 1ml syringe and use it to blow the solution 50-90 times in one step.
- Centrifuge the solution at 15000g for 10 minutes at 4°C.
- Collect the supernatant in an EP tube as cytoplasmic protein.
2. For nuclear protein extraction:
- Take the centrifugation precipitation obtained in Step 2.1.2 and add 4.0 mL of the cytoplasmic protein extraction working solution (2.0 × 10^7 cells) in an ice bath.
- Shake the solution at 4°C for 5 minutes.
- Centrifuge it at 15000g at 4°C for 10 minutes and discard the supernatant.
- Add 1.0 mL (2.0 × 10^7 cells) of a working solution for nuclear protein extraction.
By carefully following these steps, you can ensure the proper preparation of working solutions and achieve accurate protein extraction for further experiments.⁷ cells) frozen nuclear protein extract working solution, shaking at 4°C for 5 min, centrifugation at 15000g at 4°C for 10 min, the supernatant was nuclear protein, transferred into EP tube and stored. (3) The protein to be detected was stored at -70°C. For tissue samples: 1. Extraction of cytoplasmic protein (1) Add 2.0 mL /g of cytoplasmic protein extraction working solution and grind it at 4°C until it is completely dissolved; 2. (2) Oscillate for 5 min, centrifuge at 18000g for 10 min at 4°C. The supernatant was stored in the EP tube, which was cytoplasmic protein. (1) Take the precipitate in step 3.1.2 and add 4.0mL /g of cytoplasm protein extraction working solution, shake at 4°C for 5 minutes, centrifuge at 18000g at 4°C for 10 minutes, discard the supernatant; (2) Add 1.0 mL /g frozen nuclear protein extract working solution, shake at 4°C for 5 minutes, centrifuge at 18000g at 4°C for 10 minutes, the supernatant is nuclear protein, transferred into EP tube and stored; (3) The protein to be detected was stored at -70°C.