Consider the theory of ion exchange chromatography while answering the following
ID: 184895 • Letter: C
Question
Consider the theory of ion exchange chromatography while answering the following: B) why wash the column with 5 mL of 6.2 pH buffer before adding protein sample? Remember that you have equilibrated the matrix with this buffer, so it can’t be to make sure the column is at the correct pH. C) why wash the column with 5 mL of 6.2 pH buffer after addition of the protein sample? Why is this particularly important when dealing with crude cell extracts? Note that you made up he samples in 6.2 pH buffer, so your answer should not be to allow protein to get to 6.2 pH. 3) Consider the theory of ion exchange chromatography while answering the following questions. Why wash the column with 5 mL of 6.2 pH buffer before adding protein sample? Remember that you have equilibrated the matrix with this buffer, so it can't be to simply make sure the column is at the correct phH Why wash the column with 5 mL of 6.2 pH buffer after addition of the protein sample? Why is this particularly important when dealing with crude cell extracts? Note that you made up the samples in 6.2 pH buffer, so your answer should NOT that this step is to allow the protein to get to pH 6.2. Why does the hemoglobin elute first? What pH would you need to use to elute the cytochrome c from the column? Why do we elute cytochrome c with excess salt rather than raising the pH? Your answer should include the pl's of each protein (& a citation for where you obtained the data). b. c. d.Explanation / Answer
C) this is to remove unbound proteins in the sample. In crude cell extracts there will be a large number of proteins in the sample micture including the protein of your interest. Unbound unwanted proteins can be removed by washing then out with buffer of pH 6.2, which will clear the column, which has the protein or proteins of your interest bound to it.
d) The PI of hemoglobin is 6.8 and that of cytochrome C is 10.6. A protein at a pH below its pI will be positively charged. so at PH6.2 both of these are positively charged ( since the sample buffer is pH6.2) and are bound to to negatively charged cation exchange column. Once the elution starts by increasing the pH of the elution buffer at 6.8 hemoglobin elutes first since it is chargeless and is not bound to column any more and once the pH increases to near by 10.6 only chytochrome C will be eluted since it will be of no charge at that pH. Increasing salt concentration sheilds the charges on the protein and will help the protein detach from the exchanger.
b) this is to make sure that the buffer eluting from the down side of the column is also of the same pH.