I'm Dr.Ska. Today I will show you how to isolate red heart mitochondria. The key steps when isolating mitochondria from tissues are first disrupting of tissue and cells by mechanical or enzymatic treatment.
Second, differential centrifugation at low speed to remove debris and broken cells and nuclei. And third, higher speed centrifugation to collect mitochondria. The instruments and solutions were prepared and stored in the cold room in advance.
Several conditions have to be kept while isolating. Mitochondria first, thorough tissue disruption since it directly affects the yield of mitochondria. Second, keep the temperature low during all steps, but be careful of over freezing solutions, especially during centrifugation.
Third, the faster you operate, the better quality of obtained mitochondria. Animals were terminated in accordance with the UK Animal Scientific Procedure Act and all the following operations were carried out in the cold room. Prepare three beakers containing 40 mils of washing buffer.
Cool this in the ice sold bath for three to five minutes before proceeding to the next step. Make sure not to over freeze them and use immediately just after clouds of ice crystals started to appear rapidly open the thorax of the decapitated red and extract the heart. Make small incisions, and then immediately transfer the heart to the ice cold solution.
In the first beaker, squeeze the heart to remove blood and put it into the second beaker. Repeat this operation for each heart. Rapid cooling and washing of removed hearts is essential to obtain standard preparation free of hemoglobin and erythrocytes and adas.
Dry the hearts out on the filter paper. Remove all fat, clotted blood fast and artery. And pull the ventricular tissue.
Then means the pull tissue with small curved scissors on the Petri dish For five minutes, put the MIT's tissue into the tissue. Press and pass it through. After the tissue press, transfer the material into the beaker with 50 mls of washing buffer and steer.
Then filter the suspension through an island filter. Wash the men's tissue on the filter three times with the washing buffer and discard the filter. Transfer the wash tissue into 20 mils of washing buffer and place it in the ice bath on the magnetic exterior.
Add trips in solution under constant steering and start the timer on the fourth minute of incubation. Briefly homogenized the suspension portion by portion a little at a time, using a small, loosely fitted glass Teflon homogenizer. After homogenization, transfer the suspension into another beaker on a second steer.
Repeat the procedure on the ninth minute, so you do the homogenization two times in total after the second homogenization. On the 15th minute of incubation, add 10 mls of isolating buffer containing trypsin inhibitor and incubate for one More minute, Filter the suspension again through an nylon filter. But in that case, save the filtrate, transfer the rest of the tissue into the homogenizer and homogenize.
For one more minute, combine homogenate with filtrate in the centrifuge tube. Make sure the tubes are equally balanced, and do a low speed centrifugation for 15 minutes. At 600 G, carefully filter the resulting supernatant into a new centrifuge tube.
Try to avoid contamination by a pellet and centrifuge again for 15 minutes at 8, 500 G.After high speed centrifugation, discard the supernatant and rinse the pellet with a buffer trying to discard the white fluffy outer rim layer. Add one male of isolation buffer. Tilt the tube from side to side and remove the rest of the buffer.
Break up the pellet with the ula trying to avoid the red spot of erythrocytes at the bottom, resp, suspend the pellet with a small homogenizer or alternatively by gentle ine, dilute the suspension with more isolating buffer and centrifuge again for 15 minutes at 8, 500 G.Discard the S supernatant Resus. Suspend the pellet in Resus suspending buffer and centrifuge again at 8, 500 G.The resulting brown pellet contains washed intact mitochondria. Resus suspend the pellet in a very small volume of resus suspending buffer.
The lower the volume you use, the more stable the preparation of mitochondria will be. Usually one 200 microliters of the buffer per heart is enough. The The expected yield of prepared mitochondria is approximately 10 to 15 milligram of mitochondrial protein per heart.
And the respiratory control ratio on complex one substrates of such preparation should be between eight to 12. Good luck with your prep.
