ANALYTICALBIOCHEMISTRY 175,544-547 (1988)

Determination of the Molecular Weight of Proteins by Electrophoresis in Slab Gels with a Transverse Pore Gradient of Crosslinked

Polyacrylamide in the Absence of Denaturing Agents’

CLAUDIO RETAMAL* AND JORGE BABUL~

*Departamento de Biologia, and tDepartamento de Quimica, Facultad de Ciencias, Universidad de Chile, CasilIa 653, Santiago, Chile

Received May 3 1,1988

The molecular weight of proteins under nondenaturing conditions can be determined through polyacrylamide electrophoresis by comparing their relative mobilities at different gel concentra- tions with the relative mobilities of standard proteins under the same conditions (J. L. Hedrick and A. J. Smith ( 1968) Arch. Biochem. Biophys. 126,155). This work describes a procedure that eliminates the need for several gels of different acrylamide concentrations with the use of a slab gel with a transverse pore gradient of crosslinked polyacrylamide. o 1988 Academic press, IW.

KEY WORDS: gel electrophoresis; proteins/electrophoresis/molecular weights; native pro- teins/protein structure.

The rate of migration of a protein through polyacrylamide gels during electrophoresis is dependent upon its size, shape, and net charge. These features make the technique one of the most powerful and practical in the analysis and separation of proteins and other macromolecules. One of the main uses of polyacrylamide gel electrophoresis (PAGE)’ is the determination of the molecular weight of proteins in the presence of sodium dodecyl sulfate together with a thiol reagent (1). The basic goal of this approach is to convert all proteins into structures that differ only in their molecular weights. Upon binding the detergent all proteins appear to have similar shape (2,3) and an approximately constant negative charge per unit mass (4). However, when using PAGE in the presence of denatur-

’ This work was supported by grants from the Departa- mento Tkcnico de Investigaci6n (Universidad de Chile), Fondo National de Desarrollo Cientifico y Tecnol&co (Chile), and Organization of the American States.

’ Abbreviation used: PAGE, polyacrylamide gel elec- trophoresis.

ing agents it is not possible to obtain the mo- lecular weight of a native oligomeric protein. For this purpose, one may, instead, vary the sieving effect of the gel without altering the net charge and several methods have been suggested to determine the size of nondena- tured proteins using this approach (5- 12; for a review see 12). Hedrick and Smith (7) de- scribed a method where the slope of the Fer- guson plot (log of the relative mobility versus the gel concentration (5)) has a linear rela- tionship with the molecular weight of native proteins. Thus, by using a series of standard native proteins to construct such a plot it is possible to determine the molecular weight of the sample. Since the shape of the protein will also determine the rate of migration, the stan- dard proteins and the sample proteins should have the same shape, which is approximately true for most enzymes (13).

The empirical linear relationship between the relative mobilities and the molecular weight, originally proposed by Ferguson (5) and confirmed by several investigators (7-9), have been validated experimentally by Rod-

0003-2697/88 $3.00 Copyright 0 1988 by Academic Ress, Inc. All rights of reproduction in any form mserved.

544

MOLECULAR WEIGHT DETERMINATION BY ELECTROPHORESIS 545

bard and Chrambach over an extended range of pH, ionic strength, temperature, and mo- lecular weight (9). These investigators have extended Ogston’s model to predict the rela- tionship between relative mobility and mo- lecular geometry, so the determination of rel- ative mobilities under different conditions provides a mean of studying conformational changes of proteins.

In the original technique of Hedrick and Smith (7) several cylindrical gels of different acrylamide concentrations were necessary in order to construct the Ferguson plots. Now that methods are available for the prepara- tion of linear concentration gradients of acrylamide in slab gels ( 14), and electropho- resis can be performed across the gradient (6, lo), we have adapted this technique to slab gels by the use of transverse pore gradient gels of crosslinked polyacrylamide in order to de- termine the molecular weight of proteins in the absence of denaturing agents.

All reagents and proteins were from Sigma Chemical Co. PAGE was performed in a ver- tical mini-gel unit (C. B. S. Scientific Co., Model MGV- 100) according to Davis ( 15). A linear gradient of acrylamide was obtained by mixing 2 ml each of a 4 and 18% acrylamide solution with the aid of a linear gradient mixer (Biichler). The slab gel dimensions (separating gel) were 8.5 X 5.5 X 0.075 cm. After polymerization the slab gel was rotated 90” (see Fig. 1) so the acrylamide concentra- tion gradient was from left to right (18-4%) and the stacking gel solution (3% acrylamide) was added using a 15-well comb. Approxi- mately 45 pg of each standard protein was prepared in 1 mM phosphate buffer, 50 mM NaCl, pH 7, the final volume being 165 ~1, also containing 10 ~1 of 0.005% bromphenol blue, 30 ~1 of glycerol, and 30 ~1 of 1 M dithio- treitol. Aliquots of 11 rl(3 fig of protein) was loaded to each well. The running buffer was the one described by Davis (15). Electropho- resis was performed at room temperature at a constant voltage of 125 V for 90 min. The electrophoresis was terminated when the dye

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FIG. I. Slab gel with a transverse pore gradient of cross- linked polyacrylamide from 18 to 4%. The gradient was generated with a Biichler mixer using the setup shown in (A). The acrylamide concentration varied from 18% at the bottom to 4% at the top. Silicone tubing (a) sur- rounded the spacers to avoid leakage of the acrylamide solution. After polymerization of the separating gel the setup was turned 90” as shown in (B); the long spacer(b) was removed and a short one was placed on the right side of the plates so the stacking gel solution could be poured. For simplicity only one of the glass plates is shown.

reached the bottom of the gel in the 4% acryl- amide lane, the dye was marked with a fine needle, and the gel was stained with Coomas- sie blue (16) for 2 h and destained with 14% acetic acid (cf. Fig. 2). Mobilities relative to the tracking dye (R,) were calculated for each protein band at different acrylamide concen- trations assuming a linear gradient (18-4%) across the 8.5 cm of the gel. The values of R, were used to construct the Ferguson plots, 100 log (R, X 100) versus the percentage gel. The negative slopes obtained were then plot- ted against the molecular weight of each pro- tein. The linearity of acrylamide concentra- tion was verified by pouring a gel with the ad- dition of bromphenol blue in the 18% a&amide mixture chamber. After polymer- ization, a gel slice was cut from the middle of each lane and incubated for 3 h in 14% acetic acid. The absorbance at 440 nm of the super- natant liquid of each mixture was then re- corded. A linear relationship was obtained

546 RETAMAL AND BABUL

between the well number and the absorbance at 440 nm with slopes values of 0.01546 + 0.000029 (mean and standard deviation of three experiments). The assumption of lin- earity of the pore gradient is also supported by the reproducibility of the mobility and molecular weight values obtained for bovine serum albumin in six experiments. The slope values of the Ferguson plots was 6.45 -C 0.11 (means + SD). Thus, the values of M, ob- tained for a protein of 66,000 will have an av- erage deviation of +5%. As indicated by Rodbard and Chrambach (9), and attempted in this work, the precision of a Ferguson plot may be improved by increasing the number of points and the range of polyacrylamide concentration.

Figure 3 shows the relationship between the slopes of Ferguson plots against M, for four standard proteins. These data were ob- tained after electrophoresis of the proteins in slabs gels with a transverse pore gradient of crosslinked polyacrylamide, as exemplified in Fig. 2 for bovine serum albumin. Results sim-

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FIG. 2. Electrophoresis of bovine serum albumin in a slab gel with a transverse pore gradient of crosslinked polyacrylamide from 4 to 18%. The gel was prepared as described in the text using the setup of Fig. 1. A sample of albumin containing 3 pg in 11 pl was loaded in each well. Monomer (main band in each lane) and dimer spe- cies can be. observed. The thin line corresponds to the dye front at the end of the electrophoretic run.

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FIG. 3. Relationship between the slope from the plots of log R, versus the gel concentration and the molecular weight. The slope of the log R,,, versus the gel concentm- tion plot was determined for each standard protein after electrophoresis in slab gels with a transverse pore gradi- ent of crosslinked polyacrylamide from 18 to 4%. The proteins and their h4, were (1) a-la&albumin, 14,ooO; (2) carbonic anhydrase, 29,000; (3) ovalbumin, 45,000; (4) bovine serum albumin, 66,000 (monomer); (5) bovine serum albumin, 132,000 (dimer); (6) ureaq 272,000 (tri- mer); (7) urease, 545,000 (hexamer).

ilar to those of Fig. 3 were obtained when electrophoresis was performed using larger slab gels (9 X 14 X 0.15 cm) with the same acrylamide gradient. From the linear plot of Fig. 3 the molecular weight of an unknown protein can be determined. This technique is also applicable to enzymes present in impure preparations if a procedure for specific detec- tion in gels is available.

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MOLECULAR WEIGHT DETERMINATION BY ELECTROPHORESIS 547

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