इस प्रोटोकॉल एक कंकाल की मांसपेशियों से अलग mitochondria की श्वसन का अध्ययन करने की प्रक्रिया का वर्णन करता है. इस विधि Scorrano से अनुकूलित किया गया था<em> एट अल.</em> (2007). mitochondrial अलगाव की प्रक्रिया के बारे में 2 घंटे की आवश्यकता है. mitochondrial श्वसन के बारे में 1 घंटे में पूरा किया जा सकता है है.
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).
हम एक कंकाल की मांसपेशियों से व्यवहार्य mitochondria अलग प्रोटोकॉल वर्तमान. यदि उपज एक समस्या है, प्रोटोकॉल बर्फ में 30 मिनट के लिए PBS/10mM trypsin EDTA/0.01% के 5 मिलीलीटर में अलग मांसपेशी incubating द्वारा संशोधित किया जा सकता है. Trypsin…
The authors have nothing to disclose.
इस काम एफएच Andrade राष्ट्रीय नेत्र संस्थान (EY12998 R01) से एक अनुदान द्वारा समर्थित किया गया.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
95% CO2 / 5% O2 mix | Local gas company | |||
Adenosine 5′-diphosphate sodium salt | Sigma | A2754 | ||
Blue Rizla Paper | Hansatech | 890101 | ||
Bradford protein assay | Bio-Rad | 500-0006 | ||
Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) | Sigma | C2920 | ||
Centrifuge 5804R | Eppendorf | |||
Compressed nitrogen | Local gas company | |||
D-mannitol | Sigma | M9647 | ||
Ethlyene-glycol-bis-tetraacetic acid (EGTA) | Sigma | E3889 | ||
Ethylenediaminetetraacetic acid (EDTA) | Bio-Rad | 161-0728 | ||
Free fatty acid bovine serum albumin | Sigma | A8806 | ||
Glutamic acid | Sigma | G5889 | ||
HEPES sodium salt | Sigma | H7006 | ||
Isotemp 3006D | Fisher Scientific | |||
Magnesium chloride | Sigma | M8266 | ||
Male Sprague Dawley Rats | Harlan | 300-500g | ||
Malic acid | Sigma | M9138 | ||
Minifuge | ISC Bioexpress | C1301P | ||
Oligomycin | Sigma | O4876 | ||
Oxygen electrode disc | Hansatech | S1 | ||
Oxygraph | Hansatech | |||
Oxygraph Plus V1.01 Software | Hansatech | |||
pH-meter | Mettler Toledo | 1225506149 | ||
Phosphate-buffered saline (PBS) | Sigma | P4417 | ||
Potassium chloride | Sigma | P3911 | ||
Potassium phosphate | Sigma | P8416 | ||
Potter-Elvehjem homogenizers | Fisher | 08-414-14A | ||
PTFE (0.0125mm × 25mm) membrane | Hansatech | S4 | ||
SKIL 3320 drill press | Hardware store | |||
Sucrose | Sigma | S5016 |