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A protein has the following sequence: ALKJSCLKEPINWDVKLNCSLKRPILQMNLKCVGILKVN

The sequence contains three amino acids that can form a disulfide bond. However, only two of them actually make a disulfide bond in the protein. Propose a series of experiments that would allow you to determine between which two amino acids the disulfide bond occurs. Pick from: Salting out, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, SDS PAGE, affinity chromatography. HINT: do not overcomplicate the question.

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Collen Von
Collen VonLv2
28 Sep 2019

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Related questions

CHECK ALL ANSWERS THAT APPLY. THERE MAY BE MORE THAN ONE ANSWERFOR EACH QUESTION. Thank you!

You know the molecular weight of the monomeric Encprotein; however you would like to get an idea of what the pI ofthe protein is. From the following options, what is the best way toestimate that?

[ ] SDS-PAGE

[ ] Size-exclusion chromatography

[ ] 2-D gel electrophoresis

[ ] "Salt out"

Protein analysis: You are attempting to separate a60-subunit protein, Enc, from a mixture of other proteins. Eachmonomer has a molecular weight of 29 kDa. At pH 7.0, the proteinhas a net charge of -12. Which of the following options is the bestway to purify this protein?

[ ] “Salt out” via ammonium sulfate precipitation and ionexchange chromatography

[ ] Ion exchange chromatography and size-exclusionchromatography

[ ] SDS-PAGE and affinity chromatography

[ ] “Salt out” via ammonium sulfate precipitation and affinitychromatography

I have two purified samples of the enzyme PKA (ProteinKinase A). One sample is treated with cAMP (cyclic AMP), while theother other is not. The cAMP-treated sample shows 2 bands on anon-denaturing PAGE, whereas the untreated sample shows only 1band. What is the best explanation for this result?

[ ] PKA is activated by cAMP. The active enzyme would show morebands.

[ ] PAGE causes denaturation of the PKA.

[ ] cAMP unfolds PKA.

[ ] cAMP dissociates the regulatory and catalytic subunits ofPKA.

You have some protein sample and you want to quantify itusing a spectrophotometer. You take a small sample of protein andput it in a quartz cuvette, take a wavelength scan, and observeabsorbance intensity at 280 nm. Which of the following is likely tomake it so that you can analyze protein using aspectrophotometer?

[ ] Very concentrated protein sample

[ ] Proteins are composed of chiral molecules

[ ] The number of Trp and Tyr residues found in the amino acidsequence

[ ] All of the above

In the laboratory, you are studying TrbL, a 70 kD protein that causes tribbles to be furry. You isolate a 70 kD protein that you believe to be TrbL using gel filtration chromatography. When you run your sample on an SDS-PAGE gel and stain with Coomassie, you observe a single band at 70 kD. To determine the sequence of the N- terminal region of the protein, you carry out several rounds of the Edman degradation. Unfortunately, the first round suggests that you have both alanine and tyrosine present. The second round tells you that you have both tryptophan and valine present. In fact, each of the rounds of the Edman degradation suggest that two amino acids are present.

a. Assuming that your protein sequencing apparatus is functioning correctly, suggest a simple explanation for your result.

b. Give a simple experiment that will allow you to test your answer to part a.

c. How would you test your protein sequencing apparatus to be certain that it is working correctly and that your difficulties are specifically associated with your sample of 70 kD protein?

Please answer all 3 parts and give an explanation. Thank you very much! Much appreciated!

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)

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