Biochemistry Protocols: Your First Protein Purification As the newest and least
ID: 578081 • Letter: B
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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 is highly hydrated, and a concentrated ammonium sulphate solution reduces the available water very considerably.
it is possible to separate proteins from a mixture on the basis of their relative hydrophilicity by gradually increasing the concentration of ammonium sulphate.
At each stage you calculate the volume of saturated ammonium sulphate solution that will be required to achieve a given percentage saturation of your enzyme preparation, which is typically a crude tissue homogenate or perhaps a high-speed supernatant of a tissue homogenate. The ice-cold saturated solution of ammonium sulphate is added slowly to the protein solution, in an ice bath, and stirred continually. When the required amount has been added, the solution is centrifuged, and the precipitate collected, and redissolved in buffer. A higher degree of saturation with ammonium sulphate is then achieved by adding more saturated ammonium sulphate solution in the same way.
Initially you would probably use a number of wide ranges of ammonium sulphate saturation, say 0 - 50% and see whether or not your enzyme is precipitated. If it is, then you can refine the range until you achieve maximum recovery of the enzyme and maximum removal of interfering proteins.
the enzyme solution is placed in a bag of selectively permeable membrane (e.g. cellophane), immersed in a large volume of buffer that is stirred and maintained at about 4C.The membrane has pores that will permit small molecules such as ammonium and sulphate ions to cross, and hence equilibrate in the larger volume of buffer outside, while not permitting large protein molecules to cross. If the buffer is changed several times, allowing several hours each time for the ammonium sulphate to equilibrate, more or less all of it will be removed from the protein solution.
Dialysis will increase the volume of the enzyme solution, because of the initial osmotic effect of the ammonium sulphate; (this is why it is important to leave an air gap at the top of the membrane tube, to prevent it bursting)
2) A buffer solution in very general sense,makes things more stable during your measurement, and hence improves the reproducibility of any measurement. Buffer solution is resistant to pH change and also stabilize the system(here hard water) during observations/measurement.
Well so we know that water conducts electricity, but to be more precise it isn't elementally H2O which conducts it, but the dissolved ionizable salts, which is commonly present in Tap water but, distilled water, on the other hand, has a much lesser concentration of these ionizable salts, therefore your gel will run but very very slowly.
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