MBB 222 Study Guide - Final Guide: Column Chromatography, Ammonium Sulfate, Buffer Solution

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burgundycoyote951

11 Dec 2016

School

Simon Fraser University

Department

Molec Biol & Biochem

Course

MBB 222

Professor

Edgar Young

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Document Summary

Mbb222 final exam review: proteins and their techniques. There are several purification techniques to purify proteins from an enriched fraction. This is the selective aggregation or precipitation of proteins. The solubility of proteins is lowered at high salt concentrations. Takes advantage of the large size of proteins as this method separates proteins from small solutes (due to proteins being bigger) Different methods that can take advantage of several properties in proteins. A salt, usually ammonium sulfate, is added at a concentration just below the precipitation point of the protein of interest to precipitate out unwanted proteins. The unwanted insoluble proteins are said to be pelleted via centrifugation. The salt concentration of the supernatant is increased slightly to precipitate out the desired protein. The precipitate is then suspended in a physiological buffer. This method can separate proteins based on their size via a semipermeable membrane. Large molecules are retained in the dialysis bag.

Related Questions

Purification of osprase involves the following steps: homogenization, ammonium sulfate fractionation, gel filtration chromatography, ion exchange chromatography, and affinity chromatography.a) During the ammonium sulfate fractionation step you lost proteins based on their differential solubility in increasing salt concentrations, a procedure called salting out. Describe this procedure, as well as how you removed the salt afterwards.b) In the gel filtration chromatography step you collected fractions based on the presence of protein as determined by UV absorbance; five peaks of absorbance were observed, with osprase activity found in fraction 2. Explain why UV absorbance is a good way to locate proteins in solution. Sketch a plot of absorbance vs. total volume eluted. What does the fact that osprase eluted from the column in the second peak tell you?c) In a short paragraph, describe the principle of either ion exchange or affinity chromatography.

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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?

[ ] âSalt outâ via ammonium sulfate precipitation and ionexchange chromatography

[ ] Ion exchange chromatography and size-exclusionchromatography

[ ] SDS-PAGE and affinity chromatography

[ ] âSalt outâ 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

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MBB 222 Study Guide - Final Guide: Column Chromatography, Ammonium Sulfate, Buffer Solution

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