Gel Filtration Chromatography A Laboratory Experiment Jeffrey A. Hurlbut and Niels D. Schonbeck Metropolitan State College, Denver. CO 80204 Proteins can be separated by many methods, among which are gel electrophoresis, ion exchangc chromatography, affinity chromatography, and gel filtration chromatography. Few procedwes are nvailahle that readily demonstrate any of these techniques within a one- to three-hour period. We have developed a rapid, visual demonstration of protein separation by gel filtration chromatography. The procedure descrihed below separates two highly colored proteins of different molecular weight8 on Sephadex G-75 in 45 min. 'l'his time includes packing the column as well. Gel filtration chromatography (also known as gel permeation, exclusion, and molecular sieve chromatography) partitions molecules on the basis of molecular size and shape by means of a sieving proress. In normal molerular sieving, the gel network retards luge moleculen and allows small molecules to migrate through the pores. Small molecules travel farther in the gel than do large molecules. Such a pattern is observed in polyacrylamide gel electrophoresis of ~ o ~ ~ n u c l e o t i or des sodium dodecylsulfate-treated proteins, in which molecules have the same charge-to-mass ratio regardless of size. In gel filtration, however, a slight but significant change in the stationarv eel ohase causes a reversal in the norml order of elution. &&ad of a continuous gel network, the stationary phase of gel filtration is a packed bed of porous, roughly spherical beads. The resulting channels between,the beads allow passage of large molecules. Instead of being retarded by the gel network, large molecules are excluded from the gel interior and forced to travel in the channels outside the heads. Thus, large molecules have a n effectively smaller volume through which t o travel than the smaller ones, and the lareest - molecules elute first. A common form of fdtration bead, available under the trade name Sephadexa, is made from hranched polysacrharides known as dextrans, which are composed exclusively of glucosyl residues. These dextrans are treated with the crosslinkine" reagent epichlorohydrin (CH-CH-CH&I)
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to generate the porous network. Pore s u e is controlled by the concentration of crosslinkine aeent emoloved. Within the size range d e k G d by tko& large molecules which are comoletelv excluded from the eel and those small molecules which havi unrestricted access the bead interior, is the fractionation range for a particular bead pore size.' Within this molecular weight range, proteins are partitioned
on the basis of shape and s u e according to the probability of hitting the network structure rather than passing through a pore. This partitioning is sensitive enough to permit molecular weight determinations based on volume of eluant required to cause a protein to travel completely through the column (elution vol~me).~.3 In general, resolution of proteins in gel filtration is affected by height and diameter of column, bead size, volume of sample and flow rate. T o maximize the ease of performing gel filtration in the limited time of a one to three-hour period, we chose colored proteins of very different molecular weights (ferritin, yellow, 445,000 daltons, and cytochrome c, red, 12,400 daltons). Since resolution was thus not a concern, we chose column and bead parameters which maximized rate of separation. Because of time constraints in a typical lecture class, we do determination of proteins not include molecular weight ( M W ) in the procedure. ~ o w e v e istudents , should be alerted to this important application of gel filtration chromatography. A graph of log MW versus the distribution coefficient, K, generated with purified p r o t e i i of known molecular weight, gives a near-linear standard curve from which the molecular weiehts of unknown proteins can be estimated. Discussions oflthe calculation of the distribution coefficient ranee from the elementary ( K = V. - Va/Vi, where V. = elution volume of protein, Vo = column void volume, and Vi = column gel volume)=to the quite s ~ p h i s t i c a t e d . ~
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Reagents Buffer 0.05M phasphate, pH 6.9, in 0.12 M NaCl. Digpolve 3.40 g KH@OI (0.025 moles), 4.35 g KSHPOI (0.025 moles), and 7.0 g NaCl (0.12 moles) in water and dilute to 1.0 1. Protein Solutions In a small test tube. measure out 1.0me ferritin (Siwa Chemical .~ Co.. #F-4500) and d~sxolvein 0.5 ml ofbuffer. In a separate tube measure out 5.0 mg cytochrome c (Sima Chemical Co.. #C-70101 and d~ssolvein 0.5 ml buffer. To earh test tube add appnmimately 50 mg sucrose and dissolve.
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Sephadex Gel Add 5 to6 gof Sephadex G-75 (Sigma Chemical Co., #G-75-120, particle size 4CL120 w ) to 100 ml of buffer and allow to swell. The prescribed directions for swelling G-75 are to soak the beads in buffer for 24 hr at roam tem~eratureor 3 hr at 95100°C. Wehave found that I-hr making period at room temperature gives sufficient swelling of the gel for the procedure we described here. (Note that the pdyacrylamide Bio-Gel P-30or P-60 (Bio-Rod1.aboratoriesj should also work.) ~
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Wharton, D. C.. and McCarly. R. E., "Experimentsand Methods In Biochemistry." MacMilian Publishing Co., New Yo*, 1972, p. 107. Freifelder. D., "Physical Biochemlstry." W. H. Freeman and Co., S?m Francisco, 1976, pp. 185-195; Williams, B. L., and Wilson, K., "A Biologist's Guide to Principles and Techniques of Practical Biochem Istry." 2nd ed., University Park Press. Baltimore, 1981, pp. t03110. Relland, J., in "Methods In Enzymology" volume 22 (Editor: W. B. Jakoby) Academic Press, New York, 1971, pp. 287-321; Him, C. H. W.. in "M&ods in Enzymology." "01. 47 (Editors: C. H. W. Hirs and S. N. Timasheff),Academic Press, New York, 1977, pp. 97-107. Ackers, G. K.. in "The Proteins," 3rd ed. (Editors: H. Neurath and R. L. Hill), Academic Press, New York, 1975, pp. 1-92.
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Procedure Pouring the Column Place a few milliliters of the buffer intn a 3 X 40-an, muse-fdtming chromatography column. Pour the gel-buffer slurry into the column and allow it to pack under gravity. Afdter paper disc is allowed to float down to the packed gel surface to minimize disturbances during sample loading. Volume 61 Number 11
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Loading the Sample
After the students' attention has been drawn to the differencein color of the two protein samples, one solution is transferred to, and mixed with, the other by means of a disposable pipet. Conventional techniques for sample loading are adequate but time-consuming. Inatead, we apply the 1.0 ml protein sample slowly with a dispasahle pipet held in the column buffer after 1cm above the gel surface. The sucrose-spiked sample is significantly denser than buffer, and the colored protein sample can be seen layering under the buffer at the gel surface. Running the Column
Flow rates as high as 1to 2 mlhin can he used. 2.0-ml fractionsare collected. The elution profile ran be evaluated visually or quantitated by measuring absorbance at 380 nm for both peaks. Results The packed gel height is usually about 20 cm. Separation of the two proteins can he seen almost immediately as a yellow
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Journal of Chemical Education
Lining at the leading edge of the reddish sample band as i t starts down the column. Since G-75 fractionates in the 3,000-70,000 dalton range, ferritin elutes in the exclusion volume a t approximately 30 ml with a maximum ahsorhance of 0.8 a t 380 nrn.Cytochrome c elutes a t approximately 60 ml (25-30 min total running time) with a maximum ahsorhance of 0.5. As ferritin begins to elute from the column, there is a visible separation of the 2 protein hands on the column of approximately 7 cm. If this procedure is used as a laboratory experiment instead of a lecture demonstration, more care can he exercised to maximize resolution (e.g., smaller column diameter, increased attention to sample loading technique, slower flow rate, and defining gel before column packing). We have developed numerous other laboratory experiments for both hiochemistry and instrumental analysis courses. These are available upon request.
