香港留学Gel Filtration
Lin ChengyuBio 04 2010030007 Cooperator: Liu Yidi Experiment Date: 2012-03-05 & 2012-03-19 Submitting Date: 2012-04-14 1Introduction
1.1Background information
The method of gel filtration chromatography exploits the physical property of molecular
size to achieve paration. It has been of major importance in the purification of
thousands of proteins, nucleic acids, enzymes, polysaccharides, and other biomolecules.
In addition, the technique may be applied to molecular weight determination and含有一对近义词的成语
quantitative analysis of molecular interactions.
1.2Major principles
The stationary pha consists of inert particles that contain small pores of a controlled
size. Microscopic examination of a particle reveals an interior rembling a sponge.
A solution containing solutes of various molecular sizes is allowed to pass through the
column under the influence of continuous solvent flow.
Solute molecules larger than the pores cannot enter the interior of the gel beads, so they
are limited to the space between the beads. The volume of the column accessible to very
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large molecules is, therefore, greatly reduced. As a result, they are not slowed in their
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progress 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 volume
available 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 between
tho for large and small molecules.
Therefore, the order of elution of the various solute molecules is directly related to their
molecular 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 Operation
2.1Dextran-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 the
quence 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) with
elution buffer for 10 min;
(4)Adjust the flow speed to 0.5~0.6 mL / min using 10 mL graduate, fix the speed of
the 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 of
the gel column gently using dropper;
(8)Collect the solution using fraction collection, 5 min for one test tube. Meanwhile,
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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 in
each test tube at 280 nm. Also measure the total volume of each collection and部署工具
before 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 the
及格英语appliances and clamp the tongs.
2.2Standard protein
All 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 processing
3.1Raw data
Bed Volume V t = 91.5 mL
3.1.1Dextran-2000 and unknown protein
Table 1 Raw data of A and V of Dextran – 2000 and unknown protein
3.1.2Standard protein
Table 2 Raw data of A and V of standard protein
3.2Data processing
3.2.1Standard protein
Becau 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 standard
protein as shown below:
Figure 2 Elution curve of the standard protein
In 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 rum 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 with
larger relative molecular mass will be eluted faster, so that we can identify that Peak 1 corresponds with bovine rum 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 protein
From 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 protein
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3.2.2Unknown protein
Set 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 protein
Peak 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 protein
Peak 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 process
3.2.3.1Effective distribution coefficient K av
The effective distribution coefficient K av of the standard protein can be
figured out using the equation
