Description

Microsomes are spherical vesicle-like structures formed from membrane fragments following homogenization and fractionation of eukaryotic cells. The microsomal subcellular fraction is prepared by differential centrifugation and consists primarily of membranes derived from the endoplasmic reticulum (ER) and Golgi apparatus. Microsomes isolated from liver tissue are used extensively in pharmaceutical development, toxicology and environmental science to study the metabolism of drugs, organic pollutants and other xenobiotic compounds by the cytochrome P450 monooxidase (CYP) enzyme superfamily. Microsomal preparations are an affordable and convenient in vitro system for assessing Phase I biotransformation reactions, as they contain all of the xenobiotic-metabolizing CYP isozymes and the membrane-bound flavoenzymes (such as NADPH P450-Reductase and cytochrome b5) required for function of the multicomponent P450 enzyme system. BioVision’s Microsome Isolation Kit enables preparation of active microsomes in about one hour, without the need for ultracentrifugation or sucrose gradient fractionation. The kit contains sufficient reagents for 50 isolation procedures, yielding microsomes from roughly 25 grams of tissue or cultured cells.

Datasheet

FAQ

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.

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.


CITATIONS

1. Ian T. Emodin inhibits colon cancer by altering BCL-2 family proteins and cell survival pathways. Cancer Cell Int, April 2019; 31011292.
2. 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.
3. 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
4. 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
5. 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,

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