Biochemistry Protocols: Your First Protein Purification As the newest and least
ID: 191722 • Letter: B
Question
Biochemistry Protocols: Your First Protein Purification As the newest and least experience spent washing glassware and labeling test tubes. solutions for use in various laboratory pre protein. It is a citric acid cycle enzyme, citrate sy protocol for the purification, you proceed through the step student questions you about the rationale 2 dna least experienced student in a biochemistry research lab, your first few weeks are "Blassware and labeling test tubes. You then graduate to making buffers and stock o use in various laboratory procedures. Finally, you are given responsibility for purifying a citric acid cycle enzyme, citrate synthase, located in the mitochondrial matrix. Following a ication, you proceed through the steps below. As you work, a more experienced questions you about the rationale for each procedure. Supply the answers. You pick up 20 kg of beef hearts from a nea perform each step of the purification on Ice P 20 kg of beef hearts from a nearby slaughterhouse. You transport the hearts on ice, and n each step of the purification on ice or in a walk-in cold room. You homogenize the beef heart ue in a high-speed blender in a medium containing 0.2 M sucrose, buffered to a pH of 1.2. Why do you use beef heart tissue, and in such large quantity? Teort this Sure contains a lot o mitochondria, Thair ths Sue, u more mitochondria- what is the purpose of keeping the tissue cold and suspending it in 0.2 M sucrose, at pH 7.2? YRP g (t cold preserves It Ond prevents cell death. The Soluthon provides "life - like " conditions What happens to the tissue when it is homogenized? Thu a Memoranas bre o open an deuase ce It contents You subject the resulting heart homogenate, which is dense and opaque, to a series of differential centrifugation steps. What does this accomplish? This Separates out ce il components, such as nuclei mitochondria, microsomus, und nbo Soyes. You proceed with the purification using the supernatant fraction that contains mostly intact mitochondria. Next, you osmotically lyse the mitochondria. The lysate, which is less dense than the homogenate, but still opaque, consists primarily of mitochondrial membranes and internal mitochondrial contents. To this lysate you add ammonium sulfate, a highly soluble salt, to a specific concentration. You centrifuge the solution, decant the supernatant, and discard the pellet. To the supernatant, which is clearer than the lysate, you add more ammonium sulfate. Once again, you centrifuge the sample, but this time you save the pellet, because it contains the protein of interest. What is the rationale for the two-step addition of the salt? It removes on wanted proteins / components, isolates desired protein anum sulfare des alts protein Ammoni un Sultate Olsos P Solubilize the ammonium sulfate pellet containing the mitochondrial proteins and dialyze it olution. Po remove overnight against large volumes of buffered (pH 7.2) solution. Sulfat Why is ammonium sulfate not included in the dialysis buffer? why do you use the buffer solution instead of water?Explanation / Answer
1. Ammonium sulphate salt precipitates different proteins at different concentration. That is why a particular concentration of ammonium sulphate is added here to separate citrate synthetic.
2.ammonium sulphate is added to precipitate proteins. Once that has been done, ammonium sulphate needs to be removed to increase the volume of protein. This is done by removing the ammonium sulphate by dialysing the precipated protein in a buffer. Here ammonium sulphate slowly dissolves in buffer and moves out of it through selectively permeable membrane. That is why ammonium sulphate is not added in dialysing buffer.
3.Since, proteins have hydrophilic groups, if water is used in dialysing proteins, the latter will again dissolve in the former and we will have to repeat precipation step. That is the reason water is not used. Buffer keeps protein stable unlike water.
4.it tells that the first fraction would contain protein of same size and expected ones.
5 because it is quick method and does not require preparation of reagents etc. It gives results quickly whether protein are present or absent.
6.because three bands were formed, which meant three different proteins. That is why we decided to go for SDS-PAGE.
7. SDS-PAGE resulted in a single band which confirmed that the protein is pure. SDS-PAGE neutralizes the effects of charge on protein, so separates proteins solely on the basis of molecular size and yields reliable results.
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