question 3! sheet for submission. Please make sure your handwriting is legible o

Question

question 3!

sheet for submission. Please make sure your handwriting is legible on a separate sheet of paper. Staple your answers to this cover 1) (6 pts) Consult Table 3.2 in your textbook (or lecture slides) and douls in terms of AMP. Explain the trend in AG for hydrolysis resonance, and entropy of these three molecules in terms of electrostatics, ionization, three m Consider both the sign and magnitude of AG 2) (6 pts) Consider the hexapeptide (given by single letter abbreviation)y a) Draw the structure of this hexapeptide in its predominant forfo b) What is the overall charge on this at physiologicalp c) What is the overall charge on this hexapeptide in its predominant hexapeptide in its predominant form at physiological plh (7.4)? orm at plH 147 e task purifying a protein of interest (protein F) from a newly identified B, C, D, E, F and a 3) (8 pts) Dr. Eckroat has given you th bacterium for further study. After a crude cellular extract, an initial mixture of proteins A. number of small metabolites and ions is obtained. Genetic information shows that proteins A. 15, and te amino acids in size, while proteins C, E, and F are under 600 amino acids metabolic enzymes found in the cytoplasm. Protein C is suspect in size. Proteins A, D, and E are suspected ed to be a receptor embedded in the membrane. Proteins B and F are suspected to be membrane-spanning anion transport channels. The bacterium is known grow is suspected to be the in acidic soil environments and use lysine as an extracellular signaling molecule. Protein C lysine receptor. Suggest a purification protocol that would allow you to purify A from the initial misxture. I technique you would use and explain why you would use it.

Explanation / Answer

problem 3.) Purification protocol for protein A

ans. The mixture of the protein is first on the basis of the size as protein A, B, D are 1000 aminiacid in size while other are 600bp in size. Size-exclusion chromatography (also known as gel filtration) separates larger proteins from small ones since the larger molecules travel faster through the cross-linked polymer in the chromatography column. The large proteins do not fit into the pores of the polymer whereas smaller proteins do, and take longer to travel through the chromatography column, via their less direct route. Plain or cross linked agarose beads column repared first (4-6%). make sure no air is trapped inside. check for column hight by passing dH2O. equilibrate column with 2-5 volume of buffer.sample volume should be 2-5% of entire volume of column. Eluate is collected in a series of tubes separating proteins based on elution time. Gel filtration is a useful tool for concentrating a protein sample since the target protein is collected in a smaller elution volume than was initially added to the column.

By this method proteins A,B,D are separate from C,E,F. now A and D is metabolic enzyme while B is membrane spanning anion transport channels. Thus The next step is seperation of positively charge protein B be from mixture by ion exchange chromatography. anionic column prepared which contain positively charge beads. for 2 ml of protein mixture 0.2ml equilibration buffer (pH 5.5) has been added. thus when sample pass through the column protein B can not bind to the column as it is positively charge at pH 5.5. now elution buffer ( high salt and pH 8.0 ) has been pass through column. and fraction has been collected. now protein A and D has been purify from B.

A and D are metabolic protein. both has different PI value, thus 2-dimensional (2D) electrophoresis by performing isoelectric focusing and loading the resultant gel tube with proteins separated according to their pI values.The polypeptides are separated electrophoretically in polyacrylamide gel which as a pH gradient.First dimension of the 2D gel electrophoresis is established by the movement of each protein to a position, which corresponds to its isoelectric point in narrow tube.This narrow tube is again subjected to electrophoresis in a direction, which is at right angle to the direction used for iso electric focusing. In the second electrophoresis SDS is used to separate the proteins according to their sizes.The second dimension is established by the migration of the separated protein to its discrete spot on the gel.After fractionation a specific protein can be identified on the gel exposing the separated proteins to a specific antibody, which has been coupled to a label (radioactive isotope, a detectable enzyme or fluorescent dye).

thus three step sequential protein purification require to purify A from crude mixture.

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