1. What is the difference in results between a) conducting a cell permeabilizati
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Question
1. What is the difference in results between a) conducting a cell permeabilization and b) detergent extraction of a living cell to prepare the cytoskeleton?2. List the components of homogenization buffer, and what each component does.
3. Why is the nucleus removed by a low speed centrifugation?
4. Why does detergent have to be applied well above the critical micelle concentration when detergent extracting a living cell?
5. Would Tris (also called Trizma) buffer be a good buffer to be placed in "intracellular buffer"? Explain your answer.
6. After detergent extraction, how do you concentrate the soluble fraction? Explain this in detail.
7. What happens when you homogenize a membrane such as the rough endoplasmic reticulum? 1. What is the difference in results between a) conducting a cell permeabilization and b) detergent extraction of a living cell to prepare the cytoskeleton?
2. List the components of homogenization buffer, and what each component does.
3. Why is the nucleus removed by a low speed centrifugation?
4. Why does detergent have to be applied well above the critical micelle concentration when detergent extracting a living cell?
5. Would Tris (also called Trizma) buffer be a good buffer to be placed in "intracellular buffer"? Explain your answer.
6. After detergent extraction, how do you concentrate the soluble fraction? Explain this in detail.
7. What happens when you homogenize a membrane such as the rough endoplasmic reticulum? 1. What is the difference in results between a) conducting a cell permeabilization and b) detergent extraction of a living cell to prepare the cytoskeleton?
2. List the components of homogenization buffer, and what each component does.
3. Why is the nucleus removed by a low speed centrifugation?
4. Why does detergent have to be applied well above the critical micelle concentration when detergent extracting a living cell?
5. Would Tris (also called Trizma) buffer be a good buffer to be placed in "intracellular buffer"? Explain your answer.
6. After detergent extraction, how do you concentrate the soluble fraction? Explain this in detail.
7. What happens when you homogenize a membrane such as the rough endoplasmic reticulum?
Explanation / Answer
1. What is the difference in results between a) conducting a cell permeabilization and b) detergent extraction of a living cell to prepare the cytoskeleton?
Ans- conducting cell permeabilization is when organic solvents dissolve lipids from cell membranes making them permeable to antibodies. Because the organic solvents also coagulate proteins, they can be used to fix and permeabilize cells .it forms holes in the membranes. In cell permeabilization nuclear fraction and the cytoplasmic fraction are separated.
In Detergent extraction cells are separated into a cytoskeletal fraction, with all the system associated proteins, and the soluble fraction. Detergents break the protein-protein, protein-lipid and lipid-lipid associations, denature proteins and other macromolecules, prevent unspecific binding in immunochemical assays and protein crystallization.
2. List the components of homogenization buffer, and what each component does.
Ans-Lysis buffers help to break open cells, so their contents can be accessed or removed.
Buffers stabilize pH while the cells split. examples.
1.Tris-HCL stands as one of the most common chemicals for buffering at pH 8.
HEPES is another common buffer chemical in these experiments. Sodium chloride salt may also raise the ionic strength, the total concentration of solutes outside the cells. This last point has some importance since water can diffuse across cell membranes from regions of low solute concentration to regions of high solute concentration.
2.Detergents
Detergents dissolve cell membranes so the cell's contents can escape. The have and amphipathic molecular structure (i.e., molecules with one end that interacts readily with water molecules while the other hydrophobic or "water-fearing" end does not). They can dissolve fats by forming micelles, small clusters where the hydrophobic tails of the detergent molecules point inward toward the fat molecules. Common detergents include sodium dodecyl sulfate, or SDS, NP-40, and tritonX.
3. Chelating Agents
Lysis buffers typically also include chelating agents like ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA). These chemicals bind to metal ions with two positive charges (e.g., magnesium and calcium), thereby making them unavailable for other reactions. Many DNAses (proteins that chew up DNA) and proteases (proteins that slice up other proteins) need magnesium ions to function, so by depriving them of this key ingredient, EDTA and EGTA help to reduce the level of protease or DNAse activity.
3. Why is the nucleus removed by a low speed centrifugation?
Ans-low speed centrifuge is where a separation in which the components of a sample are separated on the basis of their density in a centrifuge according to the centrifugal force they experience. Samples are spun at <5000 rpm.homogenised sample is placed in an ultracentrifuge and spun in low speed - nuclei settle out, forming a pellet as those are denser.
4. Why does detergent have to be applied well above the critical micelle concentration when detergent extracting a living cell?
Ans-detergent has to be applied well above the critical micelle concentration so as to ensure that the extraction does not undergo a mistake and that there is enough critical micelle concentration rather than too little. Micellization is a critical phenomenon when considering detergent applications. Each detergent can be characterized by its critical micelle concentration (CMC); the concentration of detergent above which monomers self-assemble into non-covalent aggregates (called micelles). The CMC actually does not occur at a single concentration, but rather, over a narrow concentration range. When the total detergent concentration is below the CMC, detergent monomers are free in bulk solution. However, as more detergent is added above the CMC, all additional detergent monomers will go into micelles .
5. Would Tris (also called Trizma) buffer be a good buffer to be placed in "intracellular buffer"? Explain your answer.
Ans-if Trizma has features of a good buffer rhan it can be placed in an intracellular buffer.
1. pKa: usually between 6 and 8 desired for biological specimens.
2. Maximum solubility in water and minimum solubility in all other solvents.
3. Reduced ion effects.
4. Dissociation of buffer least influenced by buffer concentration, temperature and ionic composition.
5. Resistance to oxidation (stable).
6. Inexpensive and easy to prepare.
7. No reaction with fixation.
6. After detergent extraction, how do you concentrate the soluble fraction? Explain this in detail.
Ans-Scrape the plate and transfer to a centrifuge tube and the cytoskeleton. Then you aspirate the supernatant. Add sample piece to the pellet where the cytoskeleton is. The soluble fraction was in modified homogenization buffer. Now you concentrate it with an organize precipitation and then add sample buffer.
7. What happens when you homogenize a membrane such as the rough endoplasmic reticulum?
Ans-When tissues or cells are disrupted by homogenization, the endoplasmic reticulum and other organelles break and are floating in and unbound environment...The endoplasmisc reticulum forms small vesicles and gets binded.
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