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Product Details
| Detection Method | Western blotting, ELISA, or other assays. |
|---|---|
| Species Reactivity | Mammalian |
| Applications | Effective isolation of a highly enriched mitochondrial fraction from cytosolic fraction of mammalian cells including both apoptotic and nonapoptotic cells. |
| Features & Benefits | • Simple procedure; takes only 3-4 hours • Fast and convenient • The fractionation procedure is simple and staightforward. No ultracentrifugations are required. No toxic chemicals are involved. |
| Kit Components | • Mitochondria Extraction Buffer • 5X Cytosol Extraction Buffer • DTT (1 M) • Protease Inhibitor Cocktail |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
The Mitochondria/Cytosol Fractionation Kit provides unique formulations of reagents for effective isolation of a highly enriched mitochondrial fraction from cytosolic fraction of mammalian cells including both apoptotic and nonapoptotic cells. The enriched mitochondrial and cytosolic fractions can be used for studying apoptotic and signal transduction pathways to detect translocation of factors interested between the two fractions by Western blotting, ELISA, or other assays. Procedures are simple and easy to perform, no ultracentrifugations and toxic chemicals are involved.
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Can this product also recover cell membrane proteins? If cell membrane proteins are recovered, are they contained in the cytoplasmic fraction?
The protocol is optimized for isolating cytoplasmic and mitochondrial fractions and is not recommended for isolating membrane proteins. We do not recommend modifying any steps such as the centrifugation speed or other steps to isolate the membrane proteins. They could be present in the cytoplasmic fraction but we have never tested to determine the abundance of it in this fraction.
I want to study Cytochrome Oxidase activity in the mitochondrial fraction. What precautions should I adopt?
The Cytochrome Oxidase activity can depend a lot on multiple factors:
1. Amount of mitochondria isolated
2. Type of tissue used, (E.g..about 20-44% of rat heart samples (due to their intrinsic muscular nature) have a damaged outer mitochondrial membrane. This can lead to low Cytochrome oxidase activity readings in the samples)
3. Only freshly isolated mitochondria should be used from frozen or fresh tissues.
1. Amount of mitochondria isolated
2. Type of tissue used, (E.g..about 20-44% of rat heart samples (due to their intrinsic muscular nature) have a damaged outer mitochondrial membrane. This can lead to low Cytochrome oxidase activity readings in the samples)
3. Only freshly isolated mitochondria should be used from frozen or fresh tissues.
In which fraction are the nuclei and can they be lysed/extracted separately too with this kit?Are they in the pellet of step 7?
Yes, the nuclei are in the pellet at step 7. This pellet can be used to isolate nuclei.
After isolating the intact mitochondria, are you able to store these? If so, how and for how long are they viable for?
The mitochondrial pellet can be stored overlaid with PBS at -80C for weeks to months. But the length of storage depends on the protein of interest. Many vulnerable mitochondrial proteins (like oxidoreductases) gradually lose activity after long term storage.
I need the mitochondrial fraction to study enzymatic activity. Is this kit suitable for this?
There are no denaturing agents or heat used in the this kit. Both cytosol and mitochondria should contain fully functional native enzymes after using this kit.
Do you have some examples of the yield in mitochondrial protein amount obtained using cat# K256 with various cell lines?
The mitochondria yield depends not only on the specific cell line but how well they are proliferating and respiring. We have used this with Jurkat cells and HeLa cells and found that the yield can be between 20-100ug mito protein.
I want to use the mitochondrial fractions for co-IP and am wondering if the mitochondrial lysate can be used for this application?Are the detergents in the lysis buffer mild enough to not disrupt protein-protein interactions?
It is possible to use the fraction for co-IP applications. The extraction buffer contains >1% Triton-X, but that should not disrupt protein-protein interactions.
Will the kit work with frozen cells?
It's possible to use frozen cells with this kit.
I can see significant cytoplasmic contamination in my mitochondrial fraction. What should I do?
To minimize cytoplasmic contamination, try repeating Step 7, 2-3times. Spin the homogenate first at 700g for 10 minutes at 4C. Discard the pellet and collect the supernatant. Spin the supernatant again 2X times at 700g for 10 mins at 4C , each time collect the supernatant and discard the pellet. This will discard all unbroken cells/nucleus.
What are good markers to use for cytosolic and mitochondrial fractions?
GAPDH or alpha-tubulin are excellent markers for cytosolic fractions although some reports also refer to lactate dehydrogenase to be a superior markers for cytosolic fractions. COX-IV or VDCA1/Porin are good mitochondrial markers.
Can the kit help in isolating mitochondria from tissues?
The kit can help in isolating mitochondria and cytosolic fractions from tissues. Wash the tissue very well with ice-cold PBS and homogenize in the buffer in step B4 and proceed with the protocol.
Can the kit work on bacteria or yeast cells?
The kit has been standardized for mammalian cells only. But it can work with non-mammalian cells too. However, the end-user would need to optimize the cell lysis protocol to break the cell walls in these cell types.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
BioVisions’s assay kits expire 6 months from the date of shipment. Some components of the kit may expire sooner as mentioned in the data sheet
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
What is the purpose of a ten minute incubation at Step III-B-5 of the protocol? What does this step do to the cells?
Basically, the 1X Cytosol Extraction Buffer contains ingredients that can increases the osmotic pressure outside the cells resulting in lysis due to change in osmotic pressure. Thus, the incubation will help to prepare the cells for efficient lysis.
Ian T. Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways. Cancer Cell Int, April 2019; 31011292.
Keunjung Heo. A De Novo RAPGEF2 Variant Identified in a Sporadic Amyotrophic Lateral Sclerosis Patient Impairs Microtubule Stability and Axonal Mitochondria Distribution. Exp Neurobiol, Dec 2018; 30636905.
Wang, X. et al., (2017) Geraniin suppresses ovarian cancer growth through inhibition of NF‐κB activation and downregulation of Mcl‐1 expression, Journal of Biochemical and Molecular Toxicology, Jun.2017, 101002/jbt21929
Marni E. Cueno et al., (2017) Periodontal disease level-butyric acid putatively contributes to the ageing blood: A proposed link between periodontal diseases and the ageing process, Mechanisms of Aging and Development, 2017, 162:100-105
Ping-Yi Lin et al., (2017) Chlorella sorokiniana induces mitochondrial-mediated apoptosis in human non-small cell lung cancer cells and inhibits xenograft tumor growth in vivo, BMC Complementary and Alternative Medicine, 2017,
For more citations of this product click here
