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Mitochondrial Isolation from Skeletal Muscle

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Mitochondrial Isolation from Skeletal Muscle

This video protocol was filmed at the University of Kentucky.

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37°C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 μg) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 μM) is added to start state 3. Oligomycin (1 μM), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 μM) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR ≥4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

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Middleman haschke 'cleaned up' documents involving scam http://benjanmin.wp-site.ru/

Another delhi:Alleged intermediary guido haschke had"Cleared up"All the documents related to the chopper scam from his computer but italian investigators recovered them http://www.adultyoutubes.com/str8/my_blogs&action=add from his hard drive and additional papers he had hidden in his mother's home.

In reports, the italian detectives have termed the details found in his computer drives as"Pandora's box"When they have noted details of his appointments, his appointments with indian intermediaries, in italy and lugano and several unusual transactions including that of finmeccanica.

These documents are being used by the prosecutors in the italian court tribunale de busto arsizio to build their case in fixing required several accused including former finmeccanica chief giuseppe orsi for allegedly paying rs http://www.dailystrength.org/people/4942763/journal 360 crore in kickbacks to bag the rs 3, 600 crore put up, reports in italian daily republica recommended.

"He thought he had deleted every little thing, buried every track on the big business on the part of finmeccanica.He had 'cleaned up' all his computer system system and he felt calm, the daily said quoting an italian man, investigators' report.It said the prosecutors in the judge busto arsizio recovered the"Computer hard drives of haschke where the mid-Level had recorded the whole story about the sale of helicopters and other business,

"Documents and files that are now available of the http://demo.question2answer.org/ prosecutor of busto arsizio, eugenio fusco, who is trying to rebuild any assignments and roles of the protagonists of this 'affair', it known.The state said the"Fate of orsi is commissioned to its reading of the documents found in a suitcase that haschke had hidden in his mother's house and found those files in his computer that he thought he had cleared and reset,

Haschke is alleged to for being appointed by former finmeccanica ceo orsi for clinching the deal for agustawestland in which 51 million euros were allegedly paid as kickbacks.Orsi has denied http://beinginox.tumblr.com/post/ any kind of assistance in paying bribes for bagging the deal.

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