report
Title of experiment 3: Gel Filtration Chromatography of LDH
INTRODUCTION
Gel filtration chromatography is a type of column chromatography in which separated protein, peptides and amino acids on their molecular size. The stationary phase consists of beads containing pores. The mobile phase is the solvent that is found both around the beads and in the pores of the stationary phase matrix. As the sample is passes through the column, the molecule that are larger than the pores will not retarded by the beads and move only in the space between the beads. Thus, the larger molecule will not enter the stationary phase, staying in the mobile phase surrounding the beads. However, the molecule that smaller than the pores of the beads can diffuse further into the beads and move more slowly through the column. The small molecules can completely penetrate the pores in the beads are said to be included by the gel. The large molecules which can’t penetrate the pores are said to be excluded by the gel. Those that are excluded will pass through the column faster because the distance they have to travel is reduced and elute first whereas the smaller molecules will take a more convoluted path through the column and elute last. In this experiment, protein of interest which is LDH will be separated by using this gel filtration chromatography method.
AIMS:
1. To do the final column purification of LDH using gel filtration chromatography with Sephacryl S-200 HR.
2. To determine the activity and the absorbance of LDH in gel filtration chromatography.
3. To determine the molecular weight of the LDH.
MATERIALS AND METHODS
Please refer to page 45 to 46 of BCH204/304/3204 May 2013 session laboratory manual.
RESULTS
Volume assayed (µL)
10
Dilution used (if any)
10x
∆ Absorbance/ ∆ min
0.0221
U/mL
10.66
Total mL loaded on column
5 mL
Total units loaded on column
53.30
Table1. Summary of Concentrated/Dialyzed Sample
Table2. Data for Concentrated/Dialyzed
INTRODUCTION
Gel filtration chromatography is a type of column chromatography in which separated protein, peptides and amino acids on their molecular size. The stationary phase consists of beads containing pores. The mobile phase is the solvent that is found both around the beads and in the pores of the stationary phase matrix. As the sample is passes through the column, the molecule that are larger than the pores will not retarded by the beads and move only in the space between the beads. Thus, the larger molecule will not enter the stationary phase, staying in the mobile phase surrounding the beads. However, the molecule that smaller than the pores of the beads can diffuse further into the beads and move more slowly through the column. The small molecules can completely penetrate the pores in the beads are said to be included by the gel. The large molecules which can’t penetrate the pores are said to be excluded by the gel. Those that are excluded will pass through the column faster because the distance they have to travel is reduced and elute first whereas the smaller molecules will take a more convoluted path through the column and elute last. In this experiment, protein of interest which is LDH will be separated by using this gel filtration chromatography method.
AIMS:
1. To do the final column purification of LDH using gel filtration chromatography with Sephacryl S-200 HR.
2. To determine the activity and the absorbance of LDH in gel filtration chromatography.
3. To determine the molecular weight of the LDH.
MATERIALS AND METHODS
Please refer to page 45 to 46 of BCH204/304/3204 May 2013 session laboratory manual.
RESULTS
Volume assayed (µL)
10
Dilution used (if any)
10x
∆ Absorbance/ ∆ min
0.0221
U/mL
10.66
Total mL loaded on column
5 mL
Total units loaded on column
53.30
Table1. Summary of Concentrated/Dialyzed Sample
Table2. Data for Concentrated/Dialyzed
References: (1) Vasudevan, P.T., Palekar, A.A, and Yan, S. (2002) Optimization of lipase purification by rotating annular size-exclusion chromatography. Biocatal. Biotransform. 20, 189-199 (2) Dr. Michael Blaber (1998) Protein Purification: Gel Filtration, Affinity and Hydrophobic resins; Preparation of Resin, Plumbing Retrived from http://www.mikeblaber.org/oldwine/bch5425/lect31/lect31.htm (3) Chromatography: Introductory Theory. Biosciences. 2003. Sheffield Hallam University. Retrived from http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm