Ion Exchange Chromatography Lab Report

Abstract: Using Ion Exchange Chromatography, cellulase was purified. After purification, it was analyzed using a DNS test. The purified protein did not respond to the DNS the way it was expected to.

Introduction: Purifying proteins is an important part of biology because it can help identify the function of that protein. Once a protein’s function has been identified, it can be manipulated to see how the function would change if the protein was changed. A common way to purify a protein is through Ion Exchange Chromatography, which is where charged proteins will bind to the beads in the column to purify it from the solution (Berg JM, 2002). The purpose of this experiment is to use Ion Exchange Chromatography to purify cellulase.

Materials and Methods: An ion exchange chromatography column was obtained and set up for purification with the addition of 0.5 ml ion exchange matrix. 1 ml

equilibrium buffer was allowed to flow through the matrix before adding 1 ml of binding buffer with 1 ml of 50 mg/ml of cellulase. Then 1 ml of the wash buffer was allowed to flow through as well as 1 ml of elution buffer. All of the flow through was collected for analysis. A DNS test was run on the wash buffer and the elution buffer. To do this, four separate test tubes were made with the following: test tube 1 had 2 ml citrate buffer, 1 ml deionized water and Whatman filter paper; test tube 2 had 2 ml citrate buffer, 1 ml cellulase control and Whatman filter paper; test tube 3 had 2 ml citrate buffer, 1 ml wash buffer, 1 ml cellulase, and Whatman filter paper; test tube 4 had 2 ml citrate buffer, 1 ml elution buffer and Whatman filter paper. These four tubes were incubated at 50°C for 1 hour. After the hour, 3 ml of each tube was transferred to a different tube with 3 ml of DNS solution. One of the controls had 4.5 ml citrate buffer, 0.5 ml of 1 mg/ml glucose with 3 ml of DNS solution and the other had 3 ml citrate buffer and 3 ml DNS solution. All six of the test tubes were put in boiling water for 5 minutes. At the end of the 5 minutes, 1 ml potassium tartrate was added shortly after the tubes were put into an ice bath. Then a spectrophotometer was obtained with 7 cuvettes. 1 cuvette got 1 ml of deionized water and the other cuvettes had 1 ml from different test tubes. These were analyzed at a wavelength of 550.
Results: The absorbance numbers are as follows: test tube 1 was 0.074, test tube 2 was 2, test tube 3 was 0.037, test tube 4 was 0.077, test tube 5 was 1.08, test tube 6 was0.067. Test tubes 2 and 5 were closer to a red color than the others which were yellow. The table shows the test tube, what was in the test tubes, and the absorbance. The graph is a plot of the test tube number with their absorbance number.
550 Wavelength
Test Tube Number Contents of Test Tube Absorbance Number
1 2 ml Citrate Buffer, 1 ml DiH2O, Whatman Filter Paper 0.074
2 2 ml Citrate Buffer, 1 ml Cellulase Control, Whatman Filter Paper 2
3 2 ml Citrate Buffer, 1 ml Wash Buffer, Whatman Filter Paper 0.037
4 2 ml Citrate Buffer, 1 ml Elution Buffer, Whatman Filter Paper 0.077
5 4.5 ml Citrate Buffer, 0.5 ml Glucose 1.08
6 3 ml Citrate Buffer 0.067

Discussion: The results of the DNS test show at what absorbance of light the contents of the test tubes absorb.

Ideally the purified cellulase that was in test tube 4, which was 50 mg/ml, should have had a higher absorbance, but for unknown reasons, the cellulase has not been working all semester long. Test tube 2 had cellulase at a 1 mg/ml concentration. Next time, instead of a 1-hour incubation period at 50°C, there should be a 3-hour incubation period at 55°C. This would give the cellulase more time to break down the filter paper at a higher temperature.

Works Cited

Berg JM, T. J. (2002, January 1). Biochemistry. 5th edition. Retrieved from NCBI:

http://www.ncbi.nlm.nih.gov/books/NBK22410/