Gel filtration chromatography, also called size exclusion or molecular sieve chromatography, molecules are separated according to their size and shape. The stationary phase in these technique consists of beads of a hydrated, spongelike material containing pores that span a relatively narrow size range of molecular dimensions. If an aqueous solution containing molecules of various sizes is passed through a column containing such “molecular sieves”, the molecules that are too large to pass through the pores are excluded from the solvent volume inside the gel beads. These larger molecules therefore traverse the column more rapidly, that is, in a smaller eluent volume, than the molecules that pass through the pores.

            The molecular mass of the smallest molecule unable to penetrate the pores of a given gel is said to be the gel’s exclusion limit. The quantity is to some extent a function of molecular shape because elongated molecules, as a consequence of their higher radius of hydration,are less likely to penetrate a given gel pore than spherical molecules of the same molecular volume.

            The behavior of amoleculoe on a particular gel column can be quantitatively characterized. If Vx is the volume occupied by the gel beads and Vo, the void volume, is the volume of the solvent space surrounding the beads, then Vt, the total bed volume of the column, is simply their sum:

            The elution volume of a given solute, Ve, is the volume of the solvent required to elute the solute from the column after it has first contacted the gel. The void volume of a column is easily measured as the elution volume of a solute whose molecular mass is larger than the exclusion limit of the gel. The behavior of a particular solute on a given gel is therefore characterized by the ratio Ve/Vo, the relative elution volume, a quantity that is independent of the size of the particular column used.

            Molecules with molecular masses ranging below the exclusion limit of a gel will elute from the gel in the order of their molecular masses, with the largest eluting first. This is because the pore sizes in any gel vary over a limited range so that larger molecules have less of the gel’s interior volume available to them than smaller molecules do. This effect is the basis of gel filtration chromatography.
 

Gel filtration chromatography can be used to estimate molecular masses

            There is a linear relationship between the relative elution volume of a substance and the logarithm of its molecular mass over a considerable molecular mass range. If a plot is made for a particular gel filtration column using macromolecules of known molecular masses, the molecular mass of an unknown substance can be estimated from its position on the plot. The precision of this technique is limited by the accuracy of the underlying assumption that the known and unknown macromolecules have identical shapes. Nevertheless, gel  filtration chromatography is often used  to estimate molecular masses because it can be applied to quite impure samples,, providing that the molecule of interest can be identified, and because it can be rapidly carried out using simple equipment.
 

Reagents and Supplies

From the lockers and store room:
Sephadex G-75-50 or Sephacryl S-200 HR
0.05M Tris-HCl buffer, pH 7.5, containing 0.1M KCl
blue dextran, 1mg/mL
protein standards:
     bovine serum albumin, 5 mg/ml (Mr = 66 000)
     bovine lung aprotinin, 3 mg/ml (Mr = 6 500)
     carbonic anhydrase, 2 mg/ml (Mr = 29 000)
     cyttochrome C, 2 mg/ml  (Mr = 12 400)
unknown protein
glass rods
parafilm
cotton plug
pasteur pipettes
iron stand
clamp
pipettor and pipette tips
graduated cylinder

Things to bring:
 Double sided clip
 Dextrose tubing and regulator valve

Equipment:
 UV-Vis spectrophotometer
 Quartz cuvettes
 
 

Procedure:

A. Column Preparation
1. Set up the glass column ( a pasteur pipette ~ 1.5 x 10 cm), an iron stand and clamp, dextrose tubing and regulator valve as shown below.

2.  Fill column with water, and allow it to drain slowly. Control the rate of flow with the dextrose regulator valve or the double sided clip. Stop the flow when it is about 1/3 full.

3.  Push in a small cotton plug(0.1 – 0.3 cm thick when compressed) using a thin wire until it blocks the narrow end of the column. Repeat step 2. Set the column aside for use in Part C.

Note: To dislodge any trapped air bubbles, lightly tap the sides of the column while draining
 

B. Gel Preparation
1.  Prepare 200 ml of 50 ml Tris-HCl, pH 7.5, containing 10mM KCl. This buffer will be used to equilibrate and run the column. Keep the buffer at 4oC(refrigerator temperature) until 1 hour before use.

2. Swell Sephadex G-75-50 in water (approximately 30 ml water/g dry gel) for at least 4 hours, prferably overnight. Alternatively, the slurry can be heated in boiling water bath for 2 hours. Cool completely before use.

3. The instructor will dispense the required voloume of gel slurry into a 6 inch test tube.

4. Allow the gel to settle. Decant excess water.

5. Suspend the gel in aprroximately four volumes of equilibration buffer.

6. As the gel settles, remove the fines (slow settling particles) with a pasteur pipette. Allow the gel to settle.

7. Decant most of the supernatant, leaving a slurry consisting of 50% settled gel and 50% buffer.

Note: Steps 1 to 3 are to be done by the laboratory instructor.
 

C. Column Packing and Equilibration
1. Make a rough estimate of the column volume (total volume of liquid the column will hold).

2. Pour the slurry slowly and continuosly down a thin glass rod into the column to a bed height of 8 cm.Once you start pouring, remove the clamp and open the valve to allow the buffer to flow out of the column.

3. Replace the clamp on the column. Allow the gel to settle.

4. Open the column to allow the buffer to flow, until the buffer level is 1 cm above the gel bed. Close the column outlet.

5. To equilibrate the column, run about 2 clumn volumes of buffer, making sure that the buffer level is 1 cm above the gel bed at all times. With a pasteur pipette, replenish the buffer in the column as required.

6. Measure the column flow rate in ml/min.

WARNING! Make sure that the column never runs dry, as this will cause cracking and chanelling which in turn will cause uneven flow of sample through the gel. If this happens at any point in the experiment, the column must be repacked.

If the gel bed surface is not level, correct this by gently stirring the top of the gel with a thin glass rod or the tip of a pasteur pipette while the column is flowing.
 

D. Void Volume Determination
1. Dissolve the blue dextran in equilibration buffer  containing 5% glycerol.

2. Carefully drain the column bed until the buffer level is just barely above the gel bed.

3. Carefully apply a small volume of the blue dextran solution ( less than 2% of the total gel bed volume) onto the column. Avoid disturbing the gel bed surface. Use a pasteur pipette, and add the liquid as close to the gel bed surface as possible.

4. Allow enough buffer to drain such that all the blue dextran solution just enters the top of the gel bed surface.

5. Carefully add buffer onto the column to a depth of about 1 cm.

6. Allow the column to flow. At the same time, start collecting the eluate in 0.5 ml fractions.

7. While collecting the 0.5 ml fractions, observe the migration of blue dextran through the column.

8. When all thye blue dextran has eluted from the column, stop column flow.

9. Measure the absorbance of eluted fractions at 610 nm, and determine the void volume. (This corresponds to the absorbance peak at 610 nm).

Glycerol is added to increase the density of the solution (optional).

WARNING!Avoid disturbing the gel bed surface.

REMINDER!While running the column, constantly replenish buffer.

Note:Excessively skewed or uneven migration of the dye indicates that the column needs repacking.
 

E. Calibration of the Column
1. Dissolve individual protein standards in equilibration buffer containing 5% glycerol to make solutions with the concentrations specified above. The followign protein solutions may be mixed and run on the column simutaneously: Albumin and cytochrome C
                           Carbonic anhydrase and aprotinin

2. Apply the standard protein solutions onto the column, using a volume equivalent to that used for the blue dextran run.

3. Allow the protein solutions to enter the top of the gel bed, then start collecting the eluate in 0.5 ml fractions. Try to run the column at the same flow rate used for the void volume determination.

4. Collect about 2 column volumes of eluate.

5. Measure the absorbance offractions at 280 nm.

6. Determine Ve for each of the protein standards. (This corresponds to the absorbance peaks at 280 nm).

7. Make a calibration curve by plotting the relative molecular masses of the standard proteins against Ve/Vo using semilog paper. (Or, plot log Mr against Ve/Vo on ordianry graph paper).
 

F. Estimating Relative Molecular Mass
1. Apply the unknown protein solution onto the column, using a volume equivalent to that used for the blue dextran run.

2. Allow the protein solution to enter the top of the gel bed, then start collecting the eluate in 0.5 ml fractions (10-15 fractions should be sufficient). Try to run the column at the same flow rate used for the elution of blue dextran.

3. Measure the absorbance of fractions at 280 nm.

4. Determine Ve of unknown protein.

5. Calculate Ve/Vo for the unknown protein, and from the calibration curve, estimate its relative molecular mass.
 

G. RECYCLING THE GEL
1. Gel filtration media are expensive but are almost completely reusable. Wash the column 3-4 column volumes of buffer to elute all retained proteins.

2. Recover the gel from the column by resuspending the gel in the buffer or water, loosening the packing material by stirring the slurry, and pouring off the slurry from the column. The column may be rinsed several times for more efficient gel recovery.

Note: To prevent microbial growth, add sodium azide(NaN3) to the gel slurry.

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Chemistry 145.1
Human Genome Project and Bioinformatics
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References