Cell Mitochondria Isolation Kit

Instructions for use:
1. Prepare solutions: Melt various solutions in the kit at room temperature, place on ice immediately after melting and mix. When using for the first time, add 1.5ml PMSF (solvent) to PMSF (crystal), dissolve and mix to obtain 1.5ml 100mM PMSF. The prepared 100mM PMSF solution is stored at -20℃. If the final purpose is to prepare mitochondrial protein samples, take an appropriate amount of mitochondrial isolation reagent according to the number of samples, and add PMSF within a few minutes before the mitochondrial isolation reagent is added to the cell sample, so that the final concentration of PMSF is 1mM. At the same time, take an appropriate amount of mitochondrial lysis solution according to the number of samples, and add PMSF within a few minutes before the mitochondrial lysis solution is added to the mitochondrial sample, so that the final concentration of PMSF is 1mM.
2. Collect cells:
For adherent cells: Wash once with PBS, digest the cells with trypsin cell digestion solution (Trypsin-EDTA Solution), and collect the cells by centrifugation at 100-200g for 5-10 minutes at room temperature.
For suspended cells: directly collect cells by centrifugation.
3. Wash cells: gently resuspend the cell pellet with ice-cold PBS, take a small amount of cells for counting, and the remaining cells are 600g, centrifuged at 4℃ for 5 minutes to precipitate the cells. Discard the supernatant.
4. Pretreatment: add 1-2.5ml mitochondrial isolation reagent or mitochondrial isolation reagent with PMSF added before use to 20-50 million cells, gently suspend the cells, and place in an ice bath for 10-15 minutes.
5. Homogenization: transfer the cell suspension to a glass homogenizer of appropriate size and homogenize about 10-30 times.
6. Identification of homogenization effect: The number of homogenizations required for different cells and different homogenizers is different, and it needs to be optimized by yourself. Usually, after 10 homogenizations, take about 2 microliters of cell homogenate, add 30-50 microliters of trypan blue staining solution, mix well, and observe the proportion of trypan blue staining positive (blue) cells under a microscope. If the proportion of positive cells is less than 50%, add 5 more homogenizations. Then take samples again for trypan blue staining identification. When the positive ratio exceeds 50%, stop homogenizing and proceed to the next step. Do not over-homogenize, as over-homogenization will cause mechanical damage to mitochondria. At the same time, record the number of homogenizations for the cell. Usually, you do not need to find the number of homogenizations in subsequent experiments.
7. Centrifuge the cell homogenate at 600g and 4℃ for 10 minutes.
Note: If you need to obtain mitochondria with higher purity, you can change the centrifugation speed of this step to 1000g, and keep other centrifugation conditions unchanged; the disadvantage of obtaining higher purity mitochondria is that the mitochondrial extraction rate of the same number of cells will decrease.
8. Carefully transfer the supernatant to another centrifuge tube and centrifuge at 11,000g and 4℃ for 10 minutes.
Note: If you need to obtain mitochondria with higher purity, you can change the centrifugation speed of this step to 3500g, and keep other centrifugation conditions unchanged; the disadvantage of obtaining higher purity mitochondria is that the mitochondrial extraction rate of the same number of cells will decrease.
9. Carefully remove the supernatant. The precipitate is the isolated cell mitochondria.
Note: If you want to obtain mitochondrial-free cytoplasmic proteins, you need to collect the supernatant in this step, and be careful not to touch the precipitate when collecting the supernatant. Then centrifuge the collected supernatant at 12,000g and 4℃ for 10 minutes. The supernatant is the mitochondrial-free cytoplasmic proteins. The protein concentration of the cytoplasmic proteins can be determined using the BCA method or the Bradford method.
10. Use of mitochondria:
a. If used for the study of the function or enzyme activity of intact mitochondria, 150-200 μl of mitochondrial storage solution can be added to the mitochondrial sample obtained by initial 20-50 million cell separation to resuspend the mitochondria.
b. If used for mitochondrial protein analysis, 150-200 μl of mitochondrial lysis solution with PMSF added before use can be added to the mitochondrial sample obtained by initial 20-50 million cell separation to lyse the mitochondria. The lysed mitochondria can be used for PAGE, Western, IP, and the determination of some enzyme activities in mitochondria. The protein concentration of the lysed protein sample can be determined using the BCA method or the detergent-compatible Bradford method.
c. If used for 2D electrophoresis, treat the mitochondria with an appropriate lysis buffer for 2D electrophoresis.