Gel Filtration Experiment 2h3j1t

INDM 30020. Protein purification practical – gel filtration. Gel filtration, or molecular sieving, is a chromatographic technique for the separation of proteins in a mixture according to their molecular weight. The stationary phase consists of beads, typically of a dextran-bassed material sold under the trade name of Sephadex, which contain pores, the size of which determines the range of molecular weights that may be separated. The proteins enter the pores of the beads and their progression through the column is retarded: the smaller the protein, the slower it moves through the column. The volume of mobile phase required to elute a given protein from the column is called the elution volume (Ve), and can be used to determine the native molecular weight of a protein when compared with the elution volumes of proteins of known molecular weights. An important characteristic of a gel filtration column is the void volume, which is the volume between the beads, and can be determined by age of a molecule that has a molecular weight that is much greater than what can enter the beads (Figure).

Figure. Principles of gel filtration. Large molecules are excluded from most of the available column volume and move rapidly ahead of the solvent front.

In this experiment you will use gel filtration to separate a mixture of blue dextran (molecular weight ≅ 2,000,000) and potassium dichromate (molecular weight = 294). PROCEDURE 1. Pouring the column. Pour the slurry of Sephadex G-10 into an empty column (check that the stopcock is in the closed position) and allow the gel to settle. 2. Open the stopcock and allow the mobile phase to move through the column until it reaches the top of the gel, then close the stopcock. Do not let the column run dry. 3. Using a micropipette, gently add 100 µ l of blue dextran/potassium dichromate mixture to the top of the column. Try not to disturb the top of the gel. Open the stopcock and allow the mixture to enter the gel and close when the top of the mixture has reached the top of the gel. 4. Add mobile phase to the top of the column using a Pasteur pipette, again avoid disturbing the top of the gel. 5. Open the stopcock and collect the mobile phase in a test tube. Make sure the column does not run dry by continually adding mobile phase to the top. 6. When the blue colour has eluted from the column, use another test tube to collect the yellow fraction. Record the Ve for each of the components of the mixture. What is the void volume of your column?

AKTA prime This is a demonstration of a protein purification apparatus that is used in research labs. It is composed of pumps, a detector and an automated fraction collector. A variety of columns can be attached to the pumps depending of the type of chromatography that is to be conducted (gel-filtration, ion exchange, affinity). A schematic representation is shown in Figure 2. The column currently attached to the machine is a small gel-filtration column composing of Sephadex G-10 (the same material that you used in the first part of the practical). In research labs this column would be used to separate very large molecules, such as proteins, from very small molecules, such as salt. Thus this column could be used for buffer exchange or desalting ammonium sulphate fractions, but would not be effective at separating proteins according to size (a much longer column would be required). Buffer Chart recorder

valve Injector Pumps

Column

Detector

Fraction Collector

Waste Figure 2. Schematic diagram of the components of AKTA prime protein purification system