Product Introduction:

Nuclear extraction kits are used to isolate complete and purified nuclei from animal cells or tissues. Suitable for the preparation of animal soft tissue (liver or brain tissue) and hard tissue (muscle) as well as the nucleus of cultured cells. It has a high yield and can be used for the study of apoptosis, signaling, metabolism and proteomics. The product does not contain polluting protease and ribozyme, and the performance is stable.

Operation steps:

1. Sample processing

a. Tissue homogenate: Weigh 100~200 mg of fresh tissue such as liver, brain, myocardium, etc., rinse with PBS or normal saline, wash blood water, blot dry with filter paper, cut into pieces with scissors and put them into a small-capacity glass homogenizer. The 1.0 mL pre-cooled Lysis Buffer was added, and 50μL Reagent A was added. The tissue was ground up and down for 20 times in the ice bath at 0℃. There is unground open tissue, can be filtered with double gauze.

b. Cultured cell homogenate: digested cells, washed with PBS, centrifuged at 800 g for 5-10 min, collected cells and counted them. The cells were treated with 5 ×10⁷ cells for each extraction. 1.0 mL of ice precooled Lysis Buffer resuspension cells were added and 50μL of Reagent A was added. Cell suspension was transferred to a small-capacity glass homogenizer and cells were ground by 0℃ ice bath for 20~30 times.

2. Transfer the tissue or cell homogenate into 1.5mL centrifuge tube and centrifuge at 700g at 4℃ for 5 min. The nucleus is deposited at the bottom of the collection tube and the supernatant is discarded. Add 0.5 mL ice pre-cooled Lysis Buffer for re-suspended precipitation.

3. Take a new centrifuge tube and add 0.5 mL Medium Buffer. Absorb the re-suspension of the previous step and carefully add it into the centrifuge tube along the tube wall. Centrifuge at 4℃, 700 g, for 5min. The nucleus is deposited on the bottom of the tube.

4. Discard the supernatant and add 0.5 mL Lysis Buffer to the nuclear precipitation. Centrifuge the lysis at 1000g for 10min and discard the supernatant to obtain more pure nuclear precipitation.

5. Resuspension nuclear precipitation with 50-100μL Store Buffer or suitable reaction buffer for immediate use or -70℃ storage.

Note:

1. In order to obtain a complete nucleus, it is necessary to: first, the whole process of low temperature operation; Second, fast; Third, breaking up the cell without destroying the suborganelles is the most critical step in the preparation of the nucleus. Compared with tissue blocks, cultured cells, especially adherent cells, are more difficult to break the wall when homogenized by glass homogenizer, so small capacity glass homogenizer and tight gap pestle should be used to grind the cultured cells up and down.

2. Calculate the correct centrifugal speed with centrifugal force g, and different centrifuges can accurately calculate the centrifugal speed accordingly.

3. Western Blot and 2D-gel electrophoresis were performed, and the nucleus could be lysed by directly adding the sample buffer. Rotational speed and centrifugal force conversion: G = 1.11×(10-5)×R×[rpm]²G is the centrifugal force, generally expressed as a multiple of g (acceleration of gravity); [rpm]² : the square of the speed; R is the radius in centimeters.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.