Chem 436 Experiment 5-1 Lab Report PDF

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Lab Report for experiment 5-1. Experiment 5-1: Gel Filtration...

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Experiment 5.1 Gel Filtration

Abstract Blue dextran, cytochrome c, and K2CrO4 were separated by size using size exclusion chromatography. The Kav  of the intermediate protein was found by determining the Kav for the large protein and the small

protein. Large proteins are confined to Vo  and small proteins occupy and elute with Vt . By plugging these values into (Ve-Vo)/(Vt-Vo), to find Kav of the intermediate protein. The largest polymer was blue dextran with Kav 0. The smallest polymer was potassium chromate with Kav 1. The intermediate polymer was Cytochrome C with a calculated Kav 0.4. Introduction In this experiment, proteins were separated by size using size exclusion chromatography, which is also known as gel filtration chromatography. In Gel filtration chromatography, proteins are separated based on molecular weight and size by using a gel medium. This gel is a slurry of beads called G-75 dextran that has a fractionation range of 4-40,000 Da, and a size exclusion limit of 60,000 Da. These beads are open cross-linked polysaccharides that are porous. This filtration method causes the largest proteins to elute first, and the smallest proteins to elute last. The beads are porous and thus proteins can go through on the way down the column. The small proteins get slowed down by these pores because they can fit inside the pores and takes longer to reach the bottom of the elution column. The larger proteins can’t fit inside the pores, which causes these proteins to take a shorter route down the column. The volume in the column can be divided into two different volumes; the volume of the beads (Vi) and the volume around the beads (Vo). Adding these two volumes gives the total volume (Vt ) in the column. The relation between protein size and elution speed can be shown by Kav (Distribution coefficient). The formula for Kav is (Ve-Vo)/(Vt-Vo). Kav is inversely related to the logarithm of the size of the protein. A really large protein will have a Kav value close to 0 while a really small protein will have a value close to 1. The purpose of this experiment is to separate Blue dextran, K2CrO4, and cytochrome c by size and find the Kav value of the polymers. Methods To begin the experiment, a column was poured so that the bed of resin was 13 cm in height. The column was then equilibrated with elution buffer. A mixture of blue dextran, cytochrome c, and K2CrO4 was added to the column. The absorbance values for each fraction was then found at 430nm, 530nm, and 630nm. Results/Discussion Kav was found by determining the volume vs the max absorbance at 430nm, 530nm, and 630nm (See Fig.1). For 430nm, the max absorption was 0.343 at a fraction volume of 9mL. For, 530nm, the max absorption was 0.069 at fraction volume 6mL. For 630nm, the max absorption was 0.183 at fraction volume 4mL.

Figure 1. Graph showing the absorbance of fractions at 630, 530, and 430 nm vs. the volume of fractions in milliliters

The intermediate protein was calculated to be cytochrome C with a Kav value of 0.4. The volume values are as follows; V0 = 4mL, Ve = 6mL, Vt = 9mL, and Vi = 5mL. Polymer

MW

Log(MW)

Kav

Blue Dextran

2,000,000 Da

6.3

0

Cytochrome C

13,000 Da

4.1

0.4

K2CrO4

194 Da

2.3

1

Table 1. Table showing the MW, Log (MW), and Kav of the 3 polymers used in this experiment. The calculated Kav is highlighted in pink.

Figure 2. Linear graph of Kav vs. the log of the protein molecular weight.

Looking at the Kav data from table 2, blue dextran elutes from the column first, then cytochrome c and finally K2CrO4. Big proteins have small Kav values, while small proteins have larger Kav values. Blue dextran and potassium chromate were 0 and 1, respectively, meaning that blue dextran has the largest molecular weight and potassium chromate has the smallest. Cytochrome C has a kav of 0.4 which indicates partial penetration of cytochrome C, meaning it is the intermediate polymer (See Fig. 2).

References 1. Ninfa, Alexander J., and David P. Ballou. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. John Wiley & Sons, 2015....