Please answer #1(a-c) with work shown! Thanks For the proteins listed below indi

Question

Please answer #1(a-c) with work shown! Thanks

For the proteins listed below indicate how you would effectively separate each protein from the other proteins in that mix by answering the questions Protein name Size based on pl based on Function amino acidamino acid sequence (in sequence kDa) Binds to AMP and Ca Protein kinase, catalyzes the reaction between a protein and ATP to give ADP and a phosphorylated protein Binds to iron (Fe) Protein phosphatase, catalyzes the reaction that removes a phosphate from a phosphorylated protein Unknown but does bind to Zinc metal ions Pro1P Flx1p 215 210 7.5 7.3 Hmm1p Cnap 120 5.4 35 Unkip 42 7.8 1a. In what order do you expect the proteins to elute from the gel filtration column? List the proteins in order of first to elute to last to elute, indicating faster protein > slower protein and use a symbol to indicate proteins that would elute at the same time. 1b. When you ran the column you noticed that the order in which the proteins eluted was: Pro1p Flx1p >Hmm1p>Cnc1pUnk1p How can you explain this result? 1c. What would you do to confirm your explanation from 1b and what do you suspect is the actual size of the Cnc1p protein? How would you figure this out?

Explanation / Answer

Ans. 1a.

The proteins can be separated from the mixture by use of Gel filration Chromatography, also known as Size exclusion chromatography that works on the principle of separation on the basis of their size. In this technique, the protein sample is suspended in an aqueous solution (mobile phase), which is applied to the top of the chromatographic column, that is already filled with a matrix of porous beads (stationary phase). The smaller proteins within the sample will enter into the pores of the beads and by gravity, will travel down the column taking a long and circuitous route through the bead matrix, and thus eluted at the last. But, in contrary, the larger proteins are unable to enter the pores and thus travel down the column taking a shorter route and thus are eluted faster. Thus, in short, the protein elutes later from the column, if it is smaller. But fewer pores are accessible to the larger protein molecules and thus they elutes first.

Based on the size given in the above mentioned table, the elution order is

Pro1p ~ Flx1p > Hmm1p > Unk1p > Cnc1p (Pro1p & Flx1p are eluted first as they are larger in size but Cnc1p is eluted at the last due to smaller in size allowing them to enter into the pores of the beads)

Ans. 1b.

While running the column the elution order of the protein was noticed which differs from the presumed elution order.

Pro1p ~ Flx1p > Hmm1p > Cnc1p > Unk1p.

The Unk1p which is larger than Cnc1p eluted at the last due to possible sticking of this protein with the Stationary phase of the column. It depends on which kind of gel-filtration media is used, for example Sephadex 75 or Sephadex 200. Some gel-filtration media are weak ion exchangers and thus interact with some protein, in this case with Unk1p.

Ans. 1c.

To confirm this explanation, it would be convenient to run SDS-PAGE for the mixture and perform the LC-MS/MS technique for characterization of each bands obtained in SDS-PAGE.

To overcome the problem associated with elution, the easiest way is to increase the ionic strength of the mobile phase by addition of 0.1 - 0.2 M Arginine hydrochloride, as Arginine works very well by reducing the non-specific interactions.

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