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.
|Cat # +Size||K249-50|
|Applications||Convenient and fast isolation of microsomal fraction from animal tissues Assessment of CYP-mediated drug metabolism and xenobiotic biotransformation Protein profiling of microsomal membrane proteins by SDS-PAGE and Western blot|
|Features & Benefits||• Simple & convenient protocol • The kit provides unique formulations of ready-to-use buffers and reagents to isolate microsomes without the need for ultracentrifugation or sucrose gradient fractionation|
|Kit Components||• Homogenization Buffer • Storage Buffer • Protease Inhibitor Cocktail|
|Shipping Conditions||Gel Pack|
|USAGE||For Research Use Only! Not For Use in Humans.|
This kit includes protease inhibitors. Does this cocktail also contain phosphatase inhibitors? If not, would adding phosphatase inhibitors interfere with the assay?
The Protease Inhibitor Cocktail does not contain phosphatase inhibitors. You can add phosphatase inhibitors to the Homogenization Buffer and it will not interfere with the assay.
Can you use frozen tissues to isolate microsomes?
We recommend using fresh tissues. However, sections frozen immediately after isolation can work too. The frozen tissues should not have undergone multiple rounds of freeze and thaw.
Can I obtain not only microsome fraction but also cytosol fraction with your Microsome Isolation Kit (Cat.# K249-50)?
You can obtain cytosolic fractions from the S9 fraction. The S9 fraction was further centrifuged at >20,000 × g (preferably at 100, 000g) for 30 min to separate the microsomes and cytosolic fraction (Yoshihara and Ohta, 1998). Collect the supernatant at this step, that should be your cytosolic fraction.
Which method for the quantification of total protein amount in microsomes would you recommend?
The protein in the microsomal isolate can be quantified with a BCA assay kit.
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 your regional BioVision distributor.
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.
Tanner B. Pollock. A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells. Oxid Med Cell Longev May-19. 31198491.
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