LAB BCH 2333
Section: 10 (Thursday morning 2nd week)
Lab 2: Amino Acids and Proteins
TA: Marilyn Vera
February 16, 2012
Group 16
Graeme Jasperse 5637867
Jaime Montejo 4557893
Department of Biochemistry
University of Ottawa
Purpose:
The purpose of the first experiment is to separate the three components of a mixture of glycine, aspartic acid, and lysine by ion-exchange. This technique allows the amino acids to separate based on their pI's using a gradient of increasing pH.
The goal of the second experiment is to determine the molecular weight of an unknown protein. This is done through electrophoresis on a polyacrylamide gel. We run our unknown protein against several protein markers which is put into a gradient gel and allowed to move down its well. This works as the gradient gel allows for proteins with smaller molecular weight to travel further than the heavier proteins (Voet, D., 150). We utilize this concept to compare our unknown protein with the protein markers in order to determine its molecular weight.
R6
Table 2: Amino acids, and their corresponding pI and pH:
Amino Acid |Formula |pI* |Elution pH | |Aspartic Acid |HOOC-CH2-CH(NH2)-COOH |2.95 |4.39 | |Glycine |NH2-CH2-COOH |6.07 |5.5 | |Lysine |H2N-(CH2)4-CH(NH2)-COOH |10.07 |10.36 | |pI determined using pK scale (Voet, D., 68-69)
According to Table 2, our elution pH for aspartic acid does not match the pI of aspartic acid found in literature. This however can be related to the fact that our initial pH began at 4.30, so the acid does not bind, and it elutes quickly, thus providing us with a small peak around 4.39. The elution pH for glycine was much closer at 5.5 compared to its pI of 6.07. This tells us that the glycine is well separated from the others however it is not fully isolated, which gave us a slight decrease in the expected pH. The pH obtained from lysine was 10.36, which was very close to...