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[DESCRIPTION]
<p>This protocol is part of the FOCUS™ Mitochondria Kit <a href="https://www.protocols.io/view/Collection-of-FOCUS-Mitochondria-Kit-protocols-e8tbhwn" target="_blank">collection</a>. Please refer to the appropriate protocol depending on your application.</p>

[BEFORE_START]
<p>All buffers should be kept ice cold. Dilute appropriate volume of 3X SubCell Buffer-II to 1X with SubCell Buffer-I as needed (e.g. mix 2ml SubCell Buffer-I with 1ml SubCell Buffer-II). All centrifugation steps should be performed at 4°C.</p>

[GUIDELINES]
<p><strong>INTRODUCTION FOCUS™</strong></p>
<p>Mitochondria kit enables the fast and easy isolation of enriched mitochondrial fractions from animal cells and tissues. The majority of the isolated mitochondria obtained from this kit contain intact inner and outer membranes. This kit contains reagents for processing 50-80 preparations of 20 million cultured mammalian cells or 20-30 preps of 50-100mg tissue. The number of preparations varies depending on the protocol, preparation size and cell or tissue type.</p>
<p> </p>
<p><strong>ITEM(S) SUPPLIED (Cat. #: 786-022)</strong></p>
<table style="width: 357px; height: 178px;">
<tbody>
<tr>
<td style="width: 286.193px;"><strong>Description</strong></td>
<td style="width: 55.2841px;"><strong>Size</strong></td>
</tr>
<tr>
<td style="width: 286.193px;">SubCell Buffer-I</td>
<td style="width: 55.2841px;">60ml</td>
</tr>
<tr>
<td style="width: 286.193px;">SubCell Buffer-II [3X]</td>
<td style="width: 55.2841px;">30ml</td>
</tr>
<tr>
<td style="width: 286.193px;">SubCell Buffer-IV</td>
<td style="width: 55.2841px;">25ml</td>
</tr>
<tr>
<td style="width: 286.193px;">SubCell Buffer-V</td>
<td style="width: 55.2841px;">15ml</td>
</tr>
<tr>
<td style="width: 286.193px;">Mitochondria Storage Buffer</td>
<td style="width: 55.2841px;">10ml</td>
</tr>
<tr>
<td style="width: 286.193px;">Mitochondria Storage Component</td>
<td style="width: 55.2841px;">1 vial </td>
</tr>
</tbody>
</table>
<p> </p>
<p><strong>STORAGE CONDITION</strong></p>
<p>The kit is shipped at ambient temperature. After receiving store the kit components at 4°C except Mitochondria Storage Component at -20°C. The kit is stable for one year when stored unopened. Use aseptic techniques when handling the reagent solutions.</p>
<p> </p>
<p><strong>ADDITIONAL ITEMS REQUIRED</strong></p>
<p>Syringes and 20 gauge needles or Wheaton Dounce homogenizer, centrifuge and centrifuge tubes. Optional reagents: Delipidated BSA, Trypsin, PBS and protease inhibitor cocktail.</p>
<p> </p>
<p><strong>PREPARATION BEFORE USE</strong></p>
<p>All buffers should be kept ice cold. Dilute appropriate volume of 3X SubCell Buffer-II to 1X with SubCell Buffer-I as needed (e.g. mix 2ml SubCell Buffer-I with 1ml SubCell Buffer-II). All centrifugation steps should be performed at 4°C.</p>
<p> </p>
<p><strong>Preparation of Working Mitochondria Storage Buffer</strong></p>
<p>Pipette 0.5ml Mitochondria Storage Buffer to Mitochondria Storage Component vial. Pipette up and down a few times to dissolve all components completely. Transfer the solution of Mitochondria Storage Component to Mitochondria Storage Buffer bottle and mix well. The Working Mitochondria Storage Buffer should be kept frozen for long-term use.</p>
<p> </p>
<p><strong>Solubilization of Mitochondrial Fractions:</strong></p>
<p>The fractionated mitochondria may be solubilized in any suitable buffer consistent with downstream procedures. For IEF/2D gel electrophoresis, the mitochondria fractions may be solubilized in a chaotropic extraction buffers. G-Biosciences offers a wide selection of buffers and reagents for IEF/2D gel electrophoresis. FOCUS/Extraction Buffer-VI (Cat. # 786-233) is suitable for solubilization of mitochondria fractions.</p>

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