Fractionation of Organelles: Differential Centrifugation

In the third lab experiment I was able to isolate the Nuclei and Mitochondria from chicken liver using differential centrifugation. Though this was my first time ever using a centrifuge, I was able to learn and understand the theory behind the process. This technique is very common and was used in the experiment to separate to major organelles, the nucleus and mitochondria,  through a stepwise increase in centrifugation speed. Chicken liver and the cold fractionation buffer was homogenized and filtrated in centrifuge tubes. We used two different settings of RPM. 2500 RPM for the nuclei and 5600 for the mitochondria due to the size of the diameter, the smaller the particle the greater the RCF. 

Below is a video of the process of Cell Fractionation. . .



Comments

Post a Comment

Popular posts from this blog

The Basics of SDS - PAGE

Image
"Po lyacrylamide gels are formed by the reaction of acrylamide and bis-acrylamide ( N,N ’-methylenebisacrylamide) that results in highly cross-linked gel matrix. The gel acts as a sieve through which the proteins move in response to the electric field. Proteins contain an overall positive or negative charge; this enables the movement of a protein molecule towards the isoelectric point at which the molecule has no net charge. By denaturing the proteins and giving them a uniform negative charge, it is possible to separate them based on the size as they migrate towards the positive electrode". One aspect I truly liked about learning this technique was seeing how SDS and PAGE both combined to  use one characteristic, molecular size. The two other physical properties that are dismissed are the charge and structure due to detergents SDS  amphipathic nature. https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/gel-electrophoresis/sds-page
Read more

Buffers

Image
I n 1908, Lawrence Joseph Henderson formulated an equation which explained carbonic acid as a natural buffer. Another gentlemen , by the name of Karl Albert Hasselbalch later used Henderson's formula to work a logarithmically relation, that measures pH derivation. A buffer is simply a solution that resists changes in pH due to amounts of a base and acid as well as dilution .  Buffers are essential to everyone and specifically to my major because, as a soon to be bio-systems food and processing engineer, it is used to maintain homeostasis.  "In the food industry b uffering solutions in foods play an important role in maintaining specific pH values for optimum activity of enzymes, protein solubility, and functionality. pH may also modify the color and flavor of foods and it is a critical factor in the preservation of many processed foods. Buffering solutions are also used as reaction media in the production of food ingredients and additives. Overall pH control is a major factor ...
Read more

Determination of Protein Concentration: Spectrophotometry

Image
"There are several ways of estimating the protein concentration such as amino acid analysis following acid hydrolysis of the protein; analyzing the changes in the spectral properties of certain dyes in the presence of proteins; and spectrophotometric estimation of the proteins in near or far UV region. Although dye-binding assays and amino acid analysis following acid hydrolysis of the protein can be used for estimating the protein concentration for both pure as well as an unknown mixture of proteins; UV spectroscopic quantitation holds good for the pure proteins. If a protein is pure, UV spectroscopic quantitation is the method of choice because it is easy and less time consuming to perform; furthermore, the protein sample can be recovered back". One of the biggest obstacles as an overall whole, was the calculation part of the experiment, I have attached a video that helps better explains the process of finding the standard curve.  REFERENCES [1] Scopes, R. K. (1974) Measure...
Read more