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Description
Invent Biotechnologies MinuteTM mitochondria isolation kit is composed of optimized
buffers and protein extraction filter cartridges with 2.0 ml collection tubes. The kit is
designed to rapidly isolate native mitochondrial proteins from cultured mammalian cells
or tissues. Due to the use of protein extraction filter cartridges, the protein isolation
procedure is simple, easy and user friendly with high yield. Unlike many commercial
mitochondrial preparation kits, this kit offers wide range of starting cells (5-40
millions/sample) and intact mitochondria are isolated. The buffers are detergent and
EDTA free. A Dounce homogenizer or a tissue blender is not needed. The procedure
can be completed in about 30 min.
Applications
The kit is designed to rapidly isolate mitochondria from cultured cells or tissues for
applications such as SDS-PAGE, immunoblottings, ELISA, IP, membrane protein
structure analysis, 2-D gels, enzyme activity assays and other applications.
Buffer Formulations: Proprietary
Kit components (50 preps)
1. 15 ml buffer A
2. 30 ml buffer B
3. 50 protein extraction filter cartridges
4. 50 collection tubes with cap
5 Plastic rods (4)
6. Tissue dissociation beads
Storage: Store Buffer A and Buffer B at -20oC upon arrival.
Additional Materials Required
1 X PBS
Vortexer
Table-Top Microcentrifuge
MinuteTM Mitochondria Isolation Kit User Manual (v2) Invent Biotechnologies, Inc.
Invent Biotechnologies, Inc., 8961 Aztec Dr. Eden Prairie, MN 55347
Tel: (866)-817-7611, Fax: 952-400-6889, www.inventbiotech.com
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Important Information:
1. Read the entire procedures carefully. Thaw buffer A and buffer B completely,
invert the bottles a few times and place on ice. Chill protein extraction filter
cartridge with collection tube on ice prior to use.
2. All centrifugation steps should be performed at 4oC in a cold room or in a
refrigerated mirocentrifuge.
3. To study protein phosphorylation, phosphatase inhibitors (such as PhosStop
from Roche) should be added to buffer A prior to use. The use of protease
inhibitor cocktails is optional.
4. It is recommended to use BCA Protein Assay Kit for determination of protein
concentration (Pierce, Cat #:23227).
Mitochondrial membrane Isolation Procedures
(The rpm in this protocol is based on Eppendoff 5415C table top microcentrifuge.)
1. Thaw buffers completely, place the buffers and the filter cartridges in collection
tubs on ice.
2. Collect 5-40 X 106 cells by low speed centrifugation (500-600 X g 5 min).
3. Wash cells once with cold PBS. Remove supernatant completely and resuspend
the cell pellet in 250 μl buffer A by vortexing briefly. Incubate the cell
suspension on ice for 5-10 min and vortex vigorously for 20-30 seconds. Transfer
the cell suspension to a filter cartridge. Go to step 4.
For tissue samples place a piece of fresh/frozen tissue (20-30 mg) in a filter
cartridge. Add 250 μl buffer A to the filter and grind the tissue with a plastic rod
for one min by pushing the tissue against the surface of the filter repeatedly with
twisting force and incubate the tube on ice with cap open for 5 min. go to step 4.
If you are working with skeletal muscles add 100-120 mg tissue dissociation
beads to the filter before grinding.
Note: The presence of a small amount of un-homogenized tissue will not affect the quality of
the sample. The plastic rod is reusable. For cleaning wipe it with 75% alcohol or rinse it
with distilled water.
4. Cap the filter cartridge and centrifuge at 14,000 rpm (16,000 X g) for 30 seconds.
Discard the filter and resuspend the pellet by vortexing briefly.
Optional: For cultured cells it is recommended to resuspend the pellet in collection tube
from step 4, transfer the cell suspension to the same filter and spin at 14,000 rpm (16,000
X g) for 30 seconds. Re-passing the cells through the filter can increase the yield by 20-
30%.
5. Resuspend the pellet by vortexing and centrifuge at 3,000 rpm for 1 min, carefully
transfer the supernatant to fresh 2.0 ml tube and add 400 μl buffer B to the tube.
Mix by vortexing for 10 seconds.
MinuteTM Mitochondria Isolation Kit User Manual (v2) Invent Biotechnologies, Inc.
Invent Biotechnologies, Inc., 8961 Aztec Dr. Eden Prairie, MN 55347
Tel: (866)-817-7611, Fax: 952-400-6889, www.inventbiotech.com
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6. Centrifuge at 14,000 rpm (16,000 X g) for 10 min. Remove the supernatant
completely and resuspend the pellet in 200 μl buffer B by vigorously vortexing
for 10 seconds.
Centrifuge the tube at 10,000 rpm (7,800 X g) for 5 min. Transfer the supernatant
to a fresh 2.0 ml tube; add 1.6 ml cold PBS to the tube and centrifuge at minimum
14,000 rpm (16,000 X g) for 15 min. Discard the supernatant and save the pellet
(isolated mitochondrial proteins). Typically 5-200 μg proteins can be obtained.
Pellet of mitochondrial proteins can be dissolved in 10-200 μl detergent
containing buffers of your choice such as 0.5% Triton X-100 in calcium-free PBS.
Troubleshooting
Problem Solution
Low protein yield Increase starting cell numbers
Increase incubation time to 10 min (step3)
Low protein activity Keep lysate cold/add proteinase inhibitors
Retention of cell lysate in protein filter
cartridge after 30 seconds of centrifugation
Reduce amount of starting material or
increase centrifugation time to 2 min