1. Joshua used gel filtration chromatography to purify protein Z from crude homogenate prepared from Arabidopsis plants. He noticed that protein Z appeared to have eluted from the column in two separate peaks. Using a standard curve, he calculated the MW of protein Z eluted in the first peak to be 156 KDa, and the protein Z eluted in the second peak had the apparent MW of 78 KDa. Which one do you think is the correct MW? What does this tell you about the structure of the protein?
2. Read the following paragraph and correct all errors (4.15 points).
An unemployed high school student, Summer, has isolated a protein from a juniper tree with an overall negative charge. He performs ion exchange chromatography using a cationic resin with blue dextran to aid the proteins association with the column. As the proteins come off the column, the spec shows 4 peaks, only the first peak (wash peak) shows activity in an enzyme assay. Since all fractions associated with this peak had activity, they were pooled and total protein assayed. Total protein analysis was assayed using Bicine reagent which interacts with proteins to cause a change in absorption when measured with a spectrophotometer. The results of the total protein calculation showed that there was a modest decrease in total protein which means that there was a large amount of pure protein of his interest present in the sample.
3.You have accidentally mixed a bunch of amino acids (tryptophan, tyrosine, alanine, selenocysteine, proline, arginine) together and once your boss finds out, you will be fired on the spot. Your only hope of saving your job is to separate them before anyone finds out. You decide that the only viable option is to use ion exchange chromatography. Explain, in detail, how you would do this? (4.15 points)
4. Why should you not lose any LDH in your 20k x g spin? How does this explain an increase the fold purification of LDH in your 20k x g sample compared to your crude homogenate? (2 points)
1. Joshua used gel filtration chromatography to purify protein Z from crude homogenate prepared from Arabidopsis plants. He noticed that protein Z appeared to have eluted from the column in two separate peaks. Using a standard curve, he calculated the MW of protein Z eluted in the first peak to be 156 KDa, and the protein Z eluted in the second peak had the apparent MW of 78 KDa. Which one do you think is the correct MW? What does this tell you about the structure of the protein?
2. Read the following paragraph and correct all errors (4.15 points).
An unemployed high school student, Summer, has isolated a protein from a juniper tree with an overall negative charge. He performs ion exchange chromatography using a cationic resin with blue dextran to aid the proteins association with the column. As the proteins come off the column, the spec shows 4 peaks, only the first peak (wash peak) shows activity in an enzyme assay. Since all fractions associated with this peak had activity, they were pooled and total protein assayed. Total protein analysis was assayed using Bicine reagent which interacts with proteins to cause a change in absorption when measured with a spectrophotometer. The results of the total protein calculation showed that there was a modest decrease in total protein which means that there was a large amount of pure protein of his interest present in the sample.
3.You have accidentally mixed a bunch of amino acids (tryptophan, tyrosine, alanine, selenocysteine, proline, arginine) together and once your boss finds out, you will be fired on the spot. Your only hope of saving your job is to separate them before anyone finds out. You decide that the only viable option is to use ion exchange chromatography. Explain, in detail, how you would do this? (4.15 points)
4. Why should you not lose any LDH in your 20k x g spin? How does this explain an increase the fold purification of LDH in your 20k x g sample compared to your crude homogenate? (2 points)
