凝胶柱层析实验报告(生物化学)

Gel FiltrationLin ChengyuBio 04 2010030007 Cooperator: Liu Yidi Experiment Date: 2012-03-05 & 2012-03-19 Submitting Date: 2012-04-14 1Introduction1.1Background informationThe method of gel filtration chromatography exploits the physical property of molecularsize to achieve separation. It has been of major importance in the purification ofthousands of proteins, nucleic acids, enzymes, polysaccharides, and other biomolecules.In addition, the technique may be applied to molecular weight determination andquantitative analysis of molecular interactions.1.2Major principlesThe stationary phase consists of inert particles that contain small pores of a controlledsize. Microscopic examination of a particle reveals an interior resembling a sponge.A solution containing solutes of various molecular sizes is allowed to pass through thecolumn under the influence of continuous solvent flow.Solute molecules larger than the pores cannot enter the interior of the gel beads, so theyare limited to the space between the beads. The volume of the column accessible to verylarge molecules is, therefore, greatly reduced. As a result, they are not slowed in theirprogress through the column and elute rapidly in a single zone.Small molecules capable of diffusing in and out of the beads have a much larger volumeavailable to them. Therefore, they are delayed in their journey through the column bed.Molecules of intermediate size migrate through the column at a rate somewhere betweenthose for large and small molecules.Therefore, the order of elution of the various solute molecules is directly related to theirmolecular dimensions.Figure 1 Separation of molecules by gel filtration.A. Application of sample containing large and small molecules.B. Large molecules cannot enter gel matrix, so they move more rapidly through the column.C. Elution of the large molecules.2Experiment Operation2.1Dextran-2000 and unknown protein(1)Turn on and test the appliances, including the constant flow pump, UV monitor,graph recording meter, and fraction collector;(2)Connect the appliances using silicone tube and emulsion tube, following thesequence of constant flow pump, gel column, UV monitor and fraction collector;(3)Switch on the constant flow pump, wash the pipe (without the gel column) withelution buffer for 10 min;(4)Adjust the flow speed to 0.5~0.6 mL / min using 10 mL graduate, fix the speed ofthe constant flow pump until the experiment ends;(5)Wash the entire system using elution buffer for 30 min;(6)Measure the bed volume;(7)Load 0.5 mL sample containing dextran – 2000 and unknown protein to the top ofthe gel column gently using dropper;(8)Collect the solution using fraction collection, 5 min for one test tube. Meanwhile,using UV monitor to detect the optical density at 280 nm;(9)Using elution buffer as the blank, measure the optical density of the collection ineach test tube at 280 nm. Also measure the total volume of each collection andbefore in order to get void volume and elution volume.(10)W ash the entire system using elution buffer for 30 min before turn off all theappliances and clamp the tongs.2.2Standard proteinAll steps are the same as shown in 2.1 except:(1)Load 0.5 mL sample containing standard protein;(2)Collect the solution using fraction collection, 3 min for one test tube.3Raw data and its processing3.1Raw dataBed Volume V t = 91.5 mL3.1.1Dextran-2000 and unknown proteinTable 1 Raw data of A and V of Dextran – 2000 and unknown protein3.1.2Standard proteinTable 2 Raw data of A and V of standard protein3.2Data processing3.2.1Standard proteinBecause of the dead volume, it shall be subtracted from all the elution volume.The data shown below has already been treated.Set A280as Y axis, and V e as X axis, and plot the elution curve of standardprotein as shown below:Figure 2 Elution curve of the standard proteinIn order to decide the elution volume of each kind of protein, we need to define the center of Peak 1, 2, and 3, from left to right.The standard protein solution contains 5 mg / mL bovine serum albumin, Mr =67 000, 8 mg / mL egg albumin, Mr = 45 000, and 5 mg / mL lysozyme, Mr =14 300. According to the gel filtration theory mentioned above, molecules withlarger relative molecular mass will be eluted faster, so that we can identify that Peak 1 corresponds with bovine serum albumin, Peak 2 corresponds with egg albumin, and Peak 3 corresponds with lysozyme.Using Origin 8.0 to process the data, the data spot is hided in order to make the curve clear, the results are shown below:Figure 3 Peak data of the standard proteinFrom Figure 3, we can figure out that the elution volume for each protein is shown as in Table 3:Table 3 Elution volume of the standard protein3.2.2Unknown proteinSet A280as Y axis, and V e as X axis, and plot the elution curve of standard protein as shown below:Figure 4 Elution curve of the unknown proteinPeak 1 corresponds with dextran –2000, and Peak 2 corresponds with the unknown protein.As the process in 3.2.1, we have Figure 4 illustrating the peak data:Figure 5 Peak data of the unknown proteinPeak 1, which corresponds with dextran – 2000, reveals the void volume of the gel column which is 31.0 mL. And the elution volume of the unknown protein is 51.3 mL.3.2.3Integrate process3.2.3.1Effective distribution coefficient K avThe effective distribution coefficient K av of the standard protein can befigured out using the equationK av=(V e− V0) (V t− V0)V0 stands for the void volume, which is 31.0 ml, and V t stands for the bedvolume, which is 91.5 mL, the result is shown in Table 4.Table 4 K of the standard proteinav rTable 5 Relationship between K and Mav rFigure 6 K av– lg M r standard curveThe correlation coefficient reaches 0.99, and the linear function isK av=−0.748lg M r +3.5843.2.3.2Standard curveIn order to get the relationship between V e and lg M r, treat the data asbelow:Table 6 Relationship between V and lg MPlot V e– lg M r in Figure 6Figure 7 V e– lg M r standard curveThe correlation coefficient reaches 0.99, and the linear function isV e=−45.3lg M r+248.03.2.3.3The relative molecular mass of unknown proteinThe elution volume of the unknown protein is 51.3 mL, that is, 0.335 inK av. Using the standard curve plotted above to define its relative molecularmass.Use K av– lg M r:Figure 8 Define the M r of the unknown protein using K av– lg M r curveThe lg M r for the unknown protein is 4.34358, which indicates that therelative molecular mass is about 22 000.Use V e– lg M r to check:Figure 9 Define the M r of the unknown protein using V e– lg M r curveThe lg M r for the unknown protein is 4.34216, which indicates that therelative molecular mass is about 22 000.The results from two methods are coincident.4Results and discussion4.1ResultsThe elution curve is shown in Figure 2 and 4;The standard curve is shown in Figure 6 and 7;The relative molecular mass of the unknown protein is about 22 000.4.2Discussion4.2.1Why should the elution speed be controlled at 0.5~0.6 mL / min?If the elution speed is too high, the sample and the gel will have less time toreach the equilibrium, which leads to poorer separation.As for too low elution speed, the diffusion of the sample cannot be ignored, andwill take more time.4.2.2The influence caused by temperatureA higher temperature can help the sample and the gel reach the equilibriumfaster. In the other hand, there will be more diffusion, and cause poorerseparation.4.2.3The importance of keeping the surface smoothIf the surface is not smooth enough, it will cause the sample in the gel columndistribute asymmetry, and will not form a band in the column.5Reference【1】YU Bingbin, Introduction of Biochemistry Experiment。