Which buffer should I use for my ion exchange chromatography experiment? The two

Question

Which buffer should I use for my ion exchange chromatography experiment? The two proteins used in the experiment are cytochrome C (isoelectric point between 10.0-10.5) and ferritin (isoelectric point of 5.3). The resin used in this experiment is a strong anion exchanger known as Q-Sepharose. The buffers available with their buffering range:

Diethylamine: 9.5-11.5

Lactic ACid: 3.6-4.3

Phosphate: 6.7-7.6

Tricine: 7.8-8.9

Tris: 7.0-8.0

The picture attached shows the structure of the ion exchange group, the different buffers available with their ranges and the properties of Cytochrome C and Ferritin.

ation exchanger. A common group used in cation exchangers is the sulfonic ion, so, If the resin group is positively charged, it will hold on to negative ions and is an anion exchanger. resin this experiment is strong anion exchanger known as Q The ion exchange group is the following quaternary amine group (Q) o-CH2CHOHCH2OCH2CHOHCH2N (CH3) 3 The mobile phase in ion-exchange is usually a buffer system. The buffer pH used in this experiment is 7 as that is at least one pH unit above and below the pl of the two proteins. Also, both proteins are stable at pH 7. The type of buffer used as the mobile phase is dependent upon the type of ion exchanger and other factors. Possible buffers that can be used in this experiment are: Buffering Range Buffer Diethylamine Lactic Acid Phosphate Tricine Tris It is your job to research these buffers and choose one that would work the best in this experiment. Consider the type of ion exchanger, the pl of the proteins, the pH of the buffer, and the buffering range and type of buffer provided when making your decision. Include your choice in your pre-lab flow chart. once the buffer, resin, and pH have been chosen, the next point to consider is to how to elute the protein that has bound to the ion-exchange column resin. One of the ways to accomplish this is by adding a large excess of salt ions, such as sodium chloride. The protein bound to the column beads can exchange with similarly charged ions in the salt and the exposed protein's charges can be saturated with the salt's i of opposite charge. The proteins are then free of the beads and can elute from the column The two molecules that are being separated in this experiment are cytochrome c and is a protein which acts as an electron shuttle in the mitochondrial electron transport chain and helps to trigger apoptosis its bright when red death) mitochondria are damaged. Cytochrome c stable and colour allows for easy tracking down the length of the It has a strong absorption peak at molecular mass of cytochrome c is that stores isoelectric point 410 cellular protein range is between 10.0 Ferritin a excess iron molecules and releases it in a controlled manner when needed. It can store inside the ferritin Accessed 2010/August 12) Because of the iron core, ferritin has a broad absorption band in the ultraviolet region which ends in the visible region of the spectrum, giving a yellow to brown colour ferritin solutions, Ferritin has a molecular mass of 445 kDa and an isoelectric point of 5.3 31

Explanation / Answer

The ideal buffer used must have pH close to the pI of the protein to be separated.

Thus,

for cytochrome C, the ideal buffer to be used is diethylamine

for ferritin, the ideal buffer to be used is lactic acid

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