Question

6. You are given a mixture of proteins that you analyze by standard 2-D electrophoresis, with the following results for isoelectric points and apparent molecular weights: protein A (Mr 110,400; pl 4.60), protein B (Mr 10,100; pl 6.93), protein C (Mr 65,200; pI 7.84), and protein D (M 25,000; pI 8.15) a. After the 2-D separation, which protein will be in the upper left quadrant of the gel. given that e cathode-proximal end of the isoelectric focusing gel was on the right side of the 2-D gel? plain why, citing the physical basis of each separation step as part of your answer drawa me of the 2D-gel indicating where the electrodes positive and negative- are located in each step of the 2D gel electrophoresis) b. Which of the four proteins is the most acidie? Explain why. c. Upon gel filtration of the mixture, only 3 peaks are seen. Their molecular masses, estimated using a calibration curve with globular proteins of know Mr, correspond to 160 kDa, 65 kDa, and 10 kDa. What can you conclude if you assume that they all behave as globular proteins? Explain why d. Protein C is phosphorylated at two Tyrosine residues. After treatment of the proteins with a phosphatase, would you expect that its pls would be shifted to higher or lower values? Explain why If a mixture of B and C is subjected to carboxymethyl (CM, pKa 4.76) matrix chromatography. what optimal pH range should you use for the buffer and in what order will the proteins emerge from the column? Explain why e. If the mixture of B and C is subjected to salting in with KCI at pH 6.8, in what order would you expect the proteins to be solubilized? Explain why. f.

Answer 1

b) The four protiens which are more acidic are as follows:-

Albumin protien, Globulin protien, Glutelin protien and Gliadins protien

-  these proteins are referred to as acidic in nature because they are insoluble in dilute mineral acids and their amino acid composition shows a excess of acidic over basic amino acid residues because it reverses the charge on positively charged DNA-peptide particles. However, simply because albumin is overall negatively charged doesn't mean it has a uniform charge. There are probably pockets with positively charged or hydrophobic character