Microsomal Fraction Of Cell

Cracking the Microsomal Fraction: A Guide to Isolation and Analysis

The microsomal fraction, a heterogeneous collection of fragmented endoplasmic reticulum (ER) membranes derived from cell homogenization, holds immense significance in various biological and biomedical research fields. This subcellular fraction houses a wealth of crucial enzymes involved in diverse metabolic processes, including drug metabolism (cytochrome P450 enzymes), lipid biosynthesis, and protein synthesis and modification. Understanding its composition and functionality is critical for advancing our knowledge in drug development, toxicology, and cellular biology. However, isolating and analyzing the microsomal fraction presents unique challenges. This article addresses common issues and provides practical solutions for researchers working with this crucial cell component.

I. Challenges in Microsomal Fraction Isolation

The primary challenge lies in effectively isolating the microsomal fraction while minimizing contamination from other organelles like mitochondria, lysosomes, and nuclei. The fragility of the ER membranes during homogenization and the overlapping density of microsomes with other subcellular components necessitates careful optimization of the isolation protocol. Several factors influence the success of microsomal fraction isolation:

Homogenization Technique: Too vigorous homogenization can lead to excessive membrane fragmentation and enzyme denaturation, while insufficient homogenization leaves intact ER structures, leading to inaccurate quantification. The choice of homogenizer (e.g., Potter-Elvehjem homogenizer, sonicator) and the homogenization parameters (e.g., speed, duration, buffer composition) are crucial.

Buffer Selection: The buffer composition directly impacts membrane integrity and enzyme activity. Appropriate osmolarity, pH, and the inclusion of protease inhibitors and reducing agents are vital to prevent degradation and maintain the native structure of enzymes. For example, a Tris-buffered saline solution supplemented with protease inhibitors (like PMSF) and EDTA is commonly used.

Centrifugation Parameters: Differential centrifugation is the cornerstone of microsomal fraction isolation. Careful optimization of centrifugation speeds and durations is critical to separate microsomes from other organelles based on their sedimentation coefficients. Incorrect centrifugation parameters can lead to co-sedimentation of unwanted organelles or loss of microsomes in the supernatant.

II. Step-by-Step Microsomal Fraction Isolation

A typical microsomal fraction isolation protocol follows these steps:

1. Cell Disruption: Cells are harvested and homogenized in a pre-cooled buffer using an appropriate homogenizer. The homogenization process should be optimized to achieve maximum disruption while minimizing damage to microsomal membranes. Monitoring the homogenate under a microscope can help assess the extent of cell disruption.

2. Low-Speed Centrifugation: The homogenate is centrifuged at low speed (e.g., 1000 x g for 10 minutes) to remove nuclei and cell debris. The supernatant is carefully collected, leaving the pellet behind.

3. Mitochondrial Pellet Removal: The supernatant is further centrifuged at a higher speed (e.g., 10,000 x g for 20 minutes) to sediment mitochondria and other larger organelles. The supernatant is again carefully transferred.

4. Microsomal Pellet Isolation: The supernatant containing the microsomal fraction is subjected to ultracentrifugation at a high speed (e.g., 100,000 x g for 60 minutes). This step pellets the microsomes.

5. Pellet Resuspension: The microsomal pellet is carefully resuspended in a suitable buffer, ensuring complete dispersion. Protein concentration is determined using standard methods like the Bradford assay.

6. Quality Control: The isolated microsomal fraction's purity should be verified using appropriate techniques, such as Western blotting for specific marker proteins (e.g., cytochrome P450 enzymes for ER) or electron microscopy to visualize the membrane structures.

III. Analyzing the Microsomal Fraction

Once isolated, the microsomal fraction can be analyzed using various techniques depending on the research objectives:

Enzyme Assays: Enzyme activity measurements (e.g., cytochrome P450 activity, NADPH cytochrome c reductase activity) provide valuable information about metabolic processes.

Western Blotting: Identifies and quantifies specific proteins within the microsomal fraction.

Proteomics: Provides a comprehensive protein profile of the microsomal fraction, offering insights into its composition and functional characteristics.

Lipidomics: Characterizes the lipid composition of microsomal membranes.

IV. Troubleshooting Common Issues

Low Microsomal Yield: Insufficient homogenization, inappropriate buffer conditions, or loss during centrifugation can lead to low yields. Optimize the homogenization and centrifugation parameters and check for buffer degradation.

Contamination with other organelles: Improper centrifugation speeds or durations can result in contamination. Careful optimization of the centrifugation steps is critical.

Enzyme inactivation: Incorrect buffer pH, temperature fluctuations, or lack of protease inhibitors can lead to enzyme denaturation. Maintain cold temperatures throughout the isolation process, and ensure the buffer is properly formulated.

V. Summary

The isolation and analysis of the microsomal fraction is a crucial technique in numerous research areas. While challenging, careful optimization of homogenization, buffer composition, and centrifugation parameters, along with rigorous quality control, ensures successful isolation and accurate analysis. Understanding and addressing the challenges discussed above leads to higher-quality data and more reliable results, contributing to a deeper understanding of cellular function and drug metabolism.

FAQs

1. What is the optimal temperature for microsomal fraction isolation? The entire process should be carried out on ice or at 4°C to minimize enzyme degradation.

2. Can I freeze the microsomal fraction? Freezing can affect membrane integrity and enzyme activity. It’s recommended to use the microsomal fraction immediately after isolation or store it at -80°C in a suitable cryoprotective agent, although some loss of activity may occur.

3. How do I determine the protein concentration of the microsomal fraction? Use a standard protein assay, such as the Bradford assay or BCA assay, after resuspending the microsomal pellet.

4. What are some common marker enzymes for the microsomal fraction? Cytochrome P450 enzymes (CYP450), NADPH-cytochrome c reductase, and glucose-6-phosphatase are commonly used markers.

5. Why is the use of protease inhibitors crucial during microsomal isolation? Protease inhibitors prevent the degradation of proteins within the microsomal fraction, ensuring accurate representation of the native protein content and enzyme activity.

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Microsome - Wikipedia In cell biology, microsomes are heterogeneous vesicle-like artifacts (~20-200 nm diameter) re-formed from pieces of the endoplasmic reticulum (ER) when eukaryotic cells are broken-up in the laboratory; microsomes are not present in healthy, living cells.

Tissue subcellular fractionation and protein extraction for ... - Nature 22 Nov 2006 · The generation of a general microsomal fraction does combine many membranous fractions together, such as Golgi, ER and intracellular vesicles.

Cell Fractionation Techniques - SpringerLink Biochemical, physical, and morphological modifications of microsomal components induced by digitonin, EDTA, and pyrophosphate, J. Cell Biol. 62:717. PubMed CAS Google Scholar

Subcellular fractionation of rough microsomes - PubMed 2 Sep 2014 · When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

Microsome - an overview | ScienceDirect Topics Separate the small microsomal cell fractions. Identify the cell fractions by use of appropriate membrane markers (both positive and negative markers). Isolate and purify a specific membrane protein. Isolate and purify membrane lipids.

Microsome - an overview | ScienceDirect Topics Lysosomes play an important role in the secretion of thyroid hormones (THs), and both the mitochondria and the smooth endoplasmic reticulum (the microsomal fraction of the cell) are the sites of steroid hormone synthesis in the adrenal gland, gonads, and placenta.

Microsome isolation kit (ab206995) - Abcam Microsome Isolation Kit (ab206995) provides a convenient and fast way to isolate microsomal fractions from animal tissues for downstream applications such as assessing CYP-mediated drug metabolism and xenobiotic biotransformation, and protein profiling of microsomal membrane proteins by SDS-PAGE and western blotting.

Microsome Isolation Kit (MAK3401) - Technical Bulletin 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.

microsomal fraction - Medical Dictionary microsomal fraction a collection of tiny subcellular particles, invisible with the light MICROSCOPE, that are produced during DIFFERENTIAL CENTRIFUGATION of eukaryotic cells. Under the ELECTRON MICROSCOPE, these microsomes can be seen to consist mainly of membranes and RIBOSOMES from the ENDOPLASMIC reticulum. The microsomal fraction is thus an ...

Microsome - an overview | ScienceDirect Topics Common in vitro metabolism systems include cells (hepatocytes), the S9 fraction, microsomes, and the soluble fraction. The preparation of S9 fraction, soluble fraction, and microsomes is shown in Fig. 3.6. Microsomes are artificial structures derived from pieces of endoplasmic reticulum (ER) formed during tissue homogenization.

Microsome - an overview | ScienceDirect Topics A microsomal fraction has been isolated from all cells studied. Claude believed that microsomes possess comparable chemical properties, although the isolated particles range from 50 to 300 m µ in diameter, with the majority at 80 to 150 m/z.

cell biology - What is a microsome? - Biology Stack Exchange 29 Dec 2016 · Differential centrifugation is a technique used to separate cellular constituents into fractions. To quote, The microsomal fraction is the pellet produced when the postmitochondrial supernatant is centrifuged at 50,000g for 60 minutes. Microsomes are small vesicles enclosed by a biological membrane.

Isolation of Endoplasmic Reticulum, Mitochondria, and … Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells.

Fractionation of Cells - Molecular Biology of the Cell - NCBI … Populations of cells can be analyzed biochemically by disrupting them and fractionating their contents by ultracentrifugation. Further fractionations allow functional cell-free systems to be developed; such systems are required to determine the molecular details of …

[5] Preparation of microsomes and submicrosomal fractions: … However, different cell types contain different kinds of submicroscopic cytoplasmic inclusion, and a common centrifugation method brings down all these inclusion practice, the microsome fraction from one tissue is not the same as that from another, precisely because …

Microsomal fraction - Oxford Reference an operational term for the subcellular fraction of a homogenate of a eukaryotic cell or tissue that, on differential centrifugation, is sedimented at 105 g; the fraction from some tissues ...

ELECTRON MICROSCOPE EXAMINATION OF SUBCELLULAR FRACTIONS The microsome fraction contained 2.8 mg of phospholipids and 6.7 mg of proteins per m 2 of membrane area. After correction for ribosomal and intracisternal proteins, the latter value was lowered to 4.7 mg of membrane protein per m 2. More …

Microsomal Fraction - an overview | ScienceDirect Topics The P450 enzymes are found in a variety of organelles, and they are predominantly localized in the microsomal fraction or the mitochondria. All of the oxidative reactions performed by this group of enzymes involve the addition of an oxygen atom to a carbon-hydrogen bond:

Cell Fractionation by Centrifugation Methods | SpringerLink Our purpose is to discuss briefly some of the major difficulties likely to arise in cell fractionation, from its very first step, homogenization of tissue, to its final step, analysis of the fractions obtained and determination of their composition in terms of subcellular entities.

Quantitative profiling of differentiation-induced microsomal … In this report we describe the systematic identification and the quantitative analysis of differentiation-induced changes of proteins in the microsomal fraction of HL-60 cells using ICAT reagents, automated peptide tandem mass spectrometry, and a …

Microsomal Fraction Of Cell

Cracking the Microsomal Fraction: A Guide to Isolation and Analysis

I. Challenges in Microsomal Fraction Isolation

II. Step-by-Step Microsomal Fraction Isolation

III. Analyzing the Microsomal Fraction

IV. Troubleshooting Common Issues

V. Summary

FAQs

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