Detailed steps of centrifugation for extraction of cytoplasmic and nuclear proteins by desktop low speed freezing centrifuge- Zhejiang Hankang Medical Technology Co., Ltd.

Detailed steps of centrifugation for extraction of cytoplasmic and nuclear proteins by desktop low speed freezing centrifuge

The desktop low speed freezing centrifuge is used in the laboratory for the primary separation and extraction of biological macromolecules, sediments, etc. Its swivel head is mainly made of aluminum alloy, which is of flat type and angle type. The centrifugal tube is made of hard glass, polyethylene hard plastic and stainless steel tube. The centrifuge is equipped with a drive motor, a timer, a regulator (speed indicator) and a refrigeration system (adjustable temperature range is - 20~+40 ℃), which can replace the rotating heads with different capacities and different types of rotational speeds according to the needs of the centrifugal material.

Detailed steps of centrifugation for extraction of cytoplasmic and nuclear proteins by desktop low speed freezing centrifuge:

1. Determine the cell growth state. The cell growth state should be 80% or better:

2. Centrifuge at 1500r/min for 5min

3. Discard the supernatant, wash the cells with equal volume of cold PBS, and repeat 3 times as in step 2

4. According to the estimated precipitation amount, add 5 times of the volume of isotonicly sisbuffer into the cell particles to make the cells suspend gently and avoid foam as far as possible

5. Add lysisbuffer into the suspended cells and put it on the ice for 15min to let the cells expand, which can be checked by microscope

6. For the expanded cells in the cell experiment, add 10% IGEPALCA-630 to make the final concentration reach 0.3%, mix well, and add gently

7. Centrifuge immediately (use 1.5ml EP tube at 5500r/min for 30s for large quantities; use 50ml tube at 5500r/min for 2min for large quantities)

8. Transfer supernatant (cytoplasmic protein) to a new freezing tube

9. Suspend cells with 5 times the volume of suspended particles in isotoniclysbuffer and mix well

10. Centrifuge the cell suspension at 1500r/min for 5min, and remove the supernatant

11. Suspend particles with 2/3 times the volume of extractionbuffer

12. Place the tube on the vibrating mixer, shake and mix at 700r/min for 15min, and then at 1400r/min for 15min. The whole process should be careful to avoid foam

13. Centrifuge at 9400r/min for 15min, transfer the supernatant to a new frozen centrifuge tube after low temperature centrifugation

14.Divide into several parts and freeze them with liquid nitrogen and store them (long-term storage should be at - 80 ℃, short-term storage should be at - 20 ℃)

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