The graph below shows the changes in cytosolic protein levels (measured by the a

Question

The graph below shows the changes in cytosolic protein levels (measured by the absorbance at 595nm, y-axis) among a series of fractions collected following ion exchange chromatography using DEAE Sepharose beads. Five protein peaks can be seen in the plotted data (solid line). These peaks are the result of the elution pattern of different proteins as they are removed from the chromatography column by increasing salt concentrations in the elution buffer (see the profile of increasing salt concentration in the graph beginning with fraction #8, dashed line). Complete the table below by indicating (a) in what fraction(s) each protein peak is found, (b) the relative charges of the proteins within the five protein peaks on the graph, and (ca brief explanation of the charge values you assigned. (that is, which peaks contain proteins that are more positively charged and which peaks contain more negatively charged proteins? Use a scale in your answer where 4is most positive, followed by 3+,2+, 1+, neutral, 1-,2-,3-, and 4- (most negative). Briefly explain your reasoning for the values you assigned to each peak. ·buffer / [high-salt) Sample 100% buffers ( low-salt) Chromatography Fraction Protein Chromatographyof Proteins in Relative Charge Reasoning for Charge Values Assigned to Protein Peaks PeakFraction(s) in Protein Peak Protein Peak

Explanation / Answer

1.

DEAE sepharose is an anion-exchange resin. The resin has positive charges all over it. The buffer in which it is prepared is made in such a way the proteins to be purified bear negative charges (if we want to elute the impurities first). If the pH of the buffer is above the pI of proteins, they bear a net negative charge and bind to the resin. By increasing the ionic strength (increasing salt concentration) of the buffer or elution buffer, the proteins can be detached from the buffer.

When salt concentration is increased, more of the negatively charged chloride ions compete with the protein molecules and displace them. Displacement of proteins is dependent on the ionic strength or binding strength of the proteins to the matrix.

Protein peak

Chromatography fraction

Relative charge of proteins in protein peak

Reason for charge values

1

2

1+

The protein elutes at low salt, which indicates it does not bind to the resin much. Either the protein is positively charged or it is neutral.

2

7

Neutral

The protein fraction elutes due to absence of charge on it.

3

9.5

2-

With gradual increase in ionic strength, the negatively charged chloride ions increase and displace the negatively charged proteins

4

11.5

3-

With gradual increase in ionic strength, the negatively charged chloride ions increase and displace the negatively charged proteins

5

15

4-

With gradual increase in ionic strength, the negatively charged chloride ions increase and displace the negatively charged proteins

2. Consider 2mM NaCl stock as x and 300mM NaCl stock as y.

for 12mM NaCl solution, x = 2.9 ml and y = 0.1 ml

for 25mM NaCl solution, x = 2.76 ml and y = 0.24 ml

for 75mM NaCl solution, x = 2.26 ml, y = 0.74 ml

for 150 mM NaCl solution, x = 1.51 ml, y = 1.49 ml

Protein peak

Chromatography fraction

Relative charge of proteins in protein peak

Reason for charge values

1

2

1+

The protein elutes at low salt, which indicates it does not bind to the resin much. Either the protein is positively charged or it is neutral.

2

7

Neutral

The protein fraction elutes due to absence of charge on it.

3

9.5

2-

With gradual increase in ionic strength, the negatively charged chloride ions increase and displace the negatively charged proteins

4

11.5

3-

With gradual increase in ionic strength, the negatively charged chloride ions increase and displace the negatively charged proteins

5

15

4-

With gradual increase in ionic strength, the negatively charged chloride ions increase and displace the negatively charged proteins

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