Sep 13, 2016
Subcellular Fractionation from Skeletal or Heart Muscle Hard Tissues (FOCUS™ SubCell Kit)
- G-Biosciences
Protocol Citation: Colin Heath: Subcellular Fractionation from Skeletal or Heart Muscle Hard Tissues (FOCUS™ SubCell Kit). protocols.io https://dx.doi.org/10.17504/protocols.io.e9ibh4e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 30, 2016
Last Modified: January 08, 2018
Protocol Integer ID: 3082
Abstract
This is part of the collection of FOCUS™ SubCell protocols for the enrichment of subcellular fractions. Please refer to the appropriate protocol depending on your application.
Guidelines
INTRODUCTION
FOCUS™ SubCell kit enables the fast and easy enrichment of nuclear, mitochondrial, membrane and cytosolic fractions from animal cells. The mitochondrial fraction can be subsequently separated into heavy and light fractions by gradient centrifugation. An additional step is included to minimize contaminations of the nuclear fraction by cytoplasmic elements (see schematic on the right). The majority of mitochondria, isolated with this kit, contain intact inner and outer membranes. FOCUS™ SubCell is suitable for cultured animal cells and can be adapted for animal tissues.
ITEM(S) SUPPLIED (Cat. # 786‐260)
| Description | Size | |
| SubCell Buffer‐I | 60ml | |
| SubCell Buffer‐II [3X] | 30ml | |
| SubCell Buffer‐III | 25ml | |
| SubCell Buffer‐IV | 25ml | |
| SubCell Buffer‐V | 15ml | |
| Mitochondria Storage Buffer | 10ml | |
| Mitochondria Storage Component | 1 vial |
STORAGE CONDITION
The kit is shipped at ambient temperature. After receiving store all the kit components at 4°C except store Mitochondria Storage Component at ‐20°C. The kit is stable for one year when stored unopened. Use aseptic techniques when handling the reagent solutions.
ITEMS NEEDED BUT NOT SUPPLIED
Syringes and 20 gauge needles or Wheaton Dounce homogenizer, centrifuge and centrifuge tubes. Optional reagents: Delipidated BSA, Trypsin, PBS and protease inhibitor cocktail.
PREPARATION BEFORE USE
• 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).
NOTE: Do not dilute all 3X SubCell Buffer‐II as some steps require the 3X concentrated SubCell Buffer II.
• All centrifugation steps should be performed at 4°C.
• Preparation of Working Mitochondria Storage Buffer: 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.
NOTE: For facilitating homogenization of the hard tissue, 0.25mg/ml Trypsin should be added to 1X SubCell Buffer‐II. A concentrated BSA solution is needed to quench the proteolytic reaction after Trypsin treatment.
Solubilization of the sub‐cell fractions:
The fractionated cell organelles (nuclei or mitochondria) may be solubilized in any suitable buffer consistent with downstream procedures. For IEF/2D gel electrophoresis, the enriched 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 all pellet fractions. The soluble cytosolic fraction can be concentrated using Perfect‐ FOCUS™ kit (Cat# 786‐124). For more information visit our website at www.GBiosciences.com
Materials
MATERIALS
FOCUS™ SubCell KitG-BiosciencesCatalog #786‐260
Before start
For facilitating homogenization of the hard tissue, 0.25mg/ml Trypsin should be added to 1X SubCell Buffer‐II. A concentrated BSA solution is needed to quench the proteolytic reaction after Trypsin treatment.
Use a fresh tissue sample (obtained within one hour of sacrifice) kept on ice. Do not freeze.
Note
For facilitating homogenization of the hard tissue, 0.25mg/ml Trypsin should be added to 1X SubCell Buffer‐II. A concentrated BSA solution is needed to quench the proteolytic reaction after Trypsin treatment.
Weigh approximately 50‐100mg tissue. On a cooled glass plate, with the aid of a scalpel, mince the tissue into very small pieces.
Suspend the sample with 8 volumes of 1X SubCell Buffer‐II containing 0.25mg/ml trypsin in a 2ml centrifuge tube.
Incubate on ice for 3 minutes and then spin down the tissue for a few seconds in the centrifuge.
00:03:00
Remove the supernatant by aspiration and add 8 volumes of 1X SubCell Buffer‐II containing 0.25mg/ml Trypsin.
Incubate on ice for 20 minutes.
00:20:00
Add BSA Solution to a final concentration of 10mg/ml and mix.
Spin down the tissue at 1,000 x g for 5‐10 seconds in the centrifuge.
00:00:05
Remove the supernatant by aspiration.
Wash the pellet with 8 volumes of 1X SubCell Buffer‐II without Trypsin, and spin down the tissue for a few seconds in the centrifuge.
Remove the supernatant by aspiration and add 8 volumes of the 1X SubCell Buffer‐ II without Trypsin.
Transfer the suspension to an ice‐cold Dounce tissue homogenizer and using a loose‐fitting pestle, disaggregate the tissue with 5‐15 strokes or until the tissue sample is completely homogenized.
Using a tight‐fitting pestle, release the nuclei with 8‐10 strokes. Do not twist the pestle as nuclei shearing may occur.
Stand on ice for 2 minutes.
00:02:00
Transfer the homogenate to a centrifuge tube and leave large chunks of tissue fragments in the homogenizer to be discarded.
Note
NOTE: For further cleaning the nuclear fraction, see 'Cleaning of the Nuclear Fraction (FOCUS™ SubCell Kit)'.
Centrifuge the lysate at 700x g for 5 minutes to pellet the nuclei.
00:05:00
Note
NOTE: For further cleaning the nuclear fraction, see 'Cleaning of the Nuclear Fraction (FOCUS™ SubCell Kit)'.
Transfer the supernatant to a new tube.
Centrifuge it at 12,000xg for 10 minutes. Transfer the supernatant (post mitochondria) to a new tube. The pellet contains mitochondria.
00:10:00
Note
NOTE: To fractionate light and heavy mitochondria, and obtain more purified mitochondrial fractions, see Section 'Fractionation of Light and Heavy Mitochondria by Gradient Cushion (FOCUS™ SubCell Kit)'.
For a crude mitochondrial fraction, continue with step 19.
Suspend the mitochondrial pellet in Working Mitochondria Storage Buffer (approximately 50μl for pellet from ~100mg tissue) and keep the suspension on ice before downstream processing. The suspension may be stored on ice for up to 48 hours.
48:00:00
Note
Freezing and thawing may compromise mitochondria integrity.
Enrichment of other cell organelles: The post mitochondria supernatant from step 17-18 can be further fractionated using a variety of gradient and differential centrifugations.
Note
For example, centrifugations of the post mitochondrial supernatant at 100,000x g for 60 minutes will sediment cellular membranes. The resulting pellet is an enriched cytosolic membrane fraction and the supernatant is soluble cytosolic fraction. This cytosolic fraction may be used for further fractionation.