A. Identify the protein band(s) on the gel which are most likely to be responsib

Question

A. Identify the protein band(s) on the gel which are most likely to be responsible for the enzyme

activity? (You can give approximate sizes or indicate directly on gel with arrows, asterisks, circles, etc.) Explain your answer.

B. What predictions, if any, can you make about subunit composition of the enzyme? Explain your answer.

C. How close to being completely purified will the enzyme be if the gel filtration and isoelectric focusing are carried out as sequential steps? In other words, how many contaminating bands and how much would you still expect in a preparation in which the two procedures were carried out sequentially (i.e., either subject the 300 kDa gel filtration peak to isoelectric focusing or subject the pH 6.9 isoelectric focusing fraction to gel filtration)? You can indicate on the figure the contaminating bands or draw a figure of the expected gel.

D. Assuming that the fold-purification and yields are similar if the steps are done sequentially as compared to being done separately, what would be the

Question 20. You have partially purified an enzyme using mon-denaturing conditions. The preparation still has several bands when analyzed by SDS gel electrophoresis (Figure, lane 1). You want to identify the band(s) that are possibly the protein of interest and to completely purify the protein. You further analyze one portion of this starting material by gel filtration chromatography and the enzyme activity eludes as a single peak of activity with a molecular mass of approximately 300 kDa. You pool these fractions and determine the specificactivity and analyze the protein sample by SDS gel electrophoresis Table and Figure, lane 2). You analyze another portion of this starting material by IEC and the majority of the enzyme activity is found in a single fraction. You determine the specific activity of this fraction and analyze the protein sample by SDS gel electrophoresis Table and Figure, lane 3). Sample Specific Activity Lane 1 2 3 Starting Material 812 units/ 2 Gel Filtration 1595 units/mg 1933 units/mg IEC 1803 Legends. The figure on the right represents a Coomassie- 116 blue stained gel with 3 lanes. Each lane has 10 LLg of protein Lane 1 ginal starting material Lane 2 84 after gel filtration, Lane 3 after IEC. The figure below is a schematic showing how the samples were obtained 553 42 Starting Material (1) 300 kDa peak from gel peak from DE filtration column 2)

Explanation / Answer

Answer: A. Protein bands between 116 and 84 Kda ( ~ 100 Kda) as well as below 55Kda (~50Kda) were responsible for enzyme activity. these 2 bands were increased in concentration upon subsequent purification with the increase in speciifc activity of enzyme.

B. SInce enzyme eluted as single peak fraction in gel filteration at 300 Kda. It can be a dimeric confirmation with 2 subunits of 100 Kda and 2 subunit of 50 Kda thus making it 300 Kda in solution.

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