2. A protein was purified to homogeneity. The mass was determined to be 60 kD by
ID: 54476 • Letter: 2
Question
2. A protein was purified to homogeneity. The mass was determined to be 60 kD by gel-filtration chromatography. When gel-filtration chromatography was repeated in the presence of urea, the mass was 30 kD. When gel-filtration chromatography was repeated again in the presence of urea and -mercaptoethanol, the mass was 15 kD. a. Using this information, describe the structure of the protein. Hint: Remember how urea and -mercaptoethanol affect protein structure. b. Complete the diagram of the SDS PAGE gel that would result from analyzing the protein. Lane (column) 1 contains the molecular weight markers, whose masses are: 11; 31; 35; 45; and 120 kD. To the left of the gel, label the bands in lane 1 with their appropriate molecular weight Note that the samples were loaded at the top of the gel as pictured. Lane 2 contains the protein sample. Note the conditions under which SDS-PAGE are run.Explanation / Answer
2
a. both urea and mercaptoethanol are the denaturing agents , they help in reducing the di sulfide linkage . urea helps in denaturing the 3D structure of the proteins as the amino group of the urea interacts with the proteins destroys ,it causes the denaturation . betamercaptaoethanol removes the disulfide linkage which is present in between the cysteine residues ,so the mass of the protein is getting reduced when used urea it forms 30kda and betamercaptoethanol it breaks disulfide bonds so it reduced into half 15kda .
b, the higher molecular weight compounds cannot move faster so they will be at the top of the gel , so the reading of the 120 kda ,45kda ,35 kda , 31 kda and 11kda , first one will be higher molecular weight and last one will be smaller molecular weight , as the gel moves from the top to bottom.
3.
a ion exchange chromatography is a process which allows seperations of the ions and polar compounds based on the their affinity to the ion exchanger . cation exchage chromatography retains positively charged cations because stationary phase is negatively charged .raise in pH will decrease the protanation of the moelcules hence the molecule is less positively charged cannot bind to stationary phase . this result they cannot bind so they will elute first phe, val , lys Asp
b. primary structure of the proteins include ;
Lys -Val- Phe -Lys -Val- Asp -Val - Phe
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.