ANALYTICAL

BIOCHEMISTRY

82. 327-333 (1977)

The Use of a New Reactive Dye for Quantitation Prestained Proteins on Polyacrylamide Gels

of

HEINRICH F. BOSSHARDAND ARVED DATYNER School of Textile Technology, Unh,ersity of New South Wales, Kensington, New South Wales 2033, Australia Received December 1, 1976; accepted May 9, 1977 An evaluation of a number of commercial reactive textile dyes with regard to suitability for staining of proteins prior to polyacrylamide gel electrophoresis showed that a blue dye containing a difluorochloropyrimidyl group gave excellent quantitation. The sensitivity was as good as with other dyes suggested for the same purpose. Reaction conditions have been optimized to reach completion in 3 hr at 40°C. A simple device for slicing gels is described. The main errors responsible for experimental scatter are briefly discussed.

Poststaining of polyacrylamide gel electropherograms by immersing the gels in a concentrated solution of a suitable dye is the most common method of visualizing proteins after electrophoresis. However, the method suffers from some disadvantages because: (a) The anionic dyes, which are usually applied [e.g., Coomassie brilliant blue R250 (C.I. Acid Blue 83) (l-S)] give only semiquantitative results. This is because diffusion of the dye into the gel is slow and, unless a very long time is allowed, “ring-dyeing” of the protein is observed, i.e., the dye concentration diminishes from the periphery of the disk toward its center. As a consequence, complex formation between protein and dye is nonstoichiometric; (b) the development of the electropherogram cannot be seen during electrophoresis; and (c) after staining, time is required to remove excess dye from the gel. By using dyes capable of covalently bonding with the protein, staining can be carried out prior to electrophoresis (prestaining), and, thereby, the above disadvantages would be avoided. Griffith (6) was the first to introduce an anionic reactive dye, Remazol brilliant blue R (C.I. Reactive Blue 19) for the prestaining of several proteins. However, the limit of sensitivity was 3 pg of protein and, in addition, there was the disadvantage of introducing negative charges by the dye. The resulting change in the charge of the protein creates the possibility of a change in the electrophoretic pattern. This objection is not valid in sodium dodecyl sulfate (SDS) electrophoresis in which the electrophoretic mobility depends only on the molecular weight of the protein. 327 CopyrIght ‘T 1977 by Academic Press. Inc. All rights of reproductmn m any form reserved.

ISSN 0003-2697

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By converting Remazol brilliant blue R into the more reactive vinyl sulfone derivative,’ Datyner and Finnimore (7) used this reactive dye as a sensitive reagent for poststaining polyacrylamide gels. In addition, as will be seen below, the activated derivative Uniblue A is also suitable for prestaining purposes and shows a higher sensitivity than the Remazol brilliant blue R proposed by Griffith. To avoid a change in the charge pattern, Datyner and Finnimore (8) suggested that a cationic reactive dye would be preferable in prestaining, because, in acid gel systems, ammonium groups would be replaced by the cationic groups of the dye, provided the dye reacts largely with amino groups. These authors synthesized a dye for this purpose and were able to obtain a greater sensitivity and a quantitative relationship from as little as OS-20 pg of protein to 20 pg. However, it was necessary to react the protein with the dye for at least 3 hr at 48°C. A problem associated with the use of cationic dyes is that, above the isoelectric point of the protein, a protein-dye complex is precipitated and must be redispersed. Unfortunately , the synthesis of the cationic dye was too complicated for production on a commercial scale, and the dye is no longer available. It appears that the ultimate in prestaining has been reached with the introduction of fluorescing stains such as dansyl chloride (9)) fluorescamine (lo- 13), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) (13,14). Protein quantities as small as 1 ng can be detected with MDPF, which is said not to have the disadvantage of the former two stains, the fluorescence of which diminishes with time. Ultraviolet radiation must be used to visualize the stains, and, for quantitative work, expensive equipment is required. In addition, due to a change in the charge of the protein by the fluorescent stains, the electrophoretic pattern may also be altered. Therefore, it seemed of interest to carry out an evaluation of existing anionic reactive textile dyes with respect to their suitability for prestaining proteins for routine work. The work pointed to the dye Drimarene brilliant blue K-BL (C.I. Reactive Blue 114) which looked promising. The structure of this dye has not been revealed by the manufacturer, but it is believed to contain the reactive 2,4-difluoro-5chloropyrimidyl group, which is more reactive than the vinyl sulfone group of Uniblue A or the sulfuric acid ester of the p-hydroxyethylsulfone group of Remazol brilliant blue R that had been applied by previous workers (6,15). EXPERIMENTAL Materials Dyes. Uniblue A (U) (Unisearch Ltd., Kensington 2033, Australia) and Drimarene brilliant blue K-BL (D) (Sandoz A. G., Basel, Switzerland) were used without any further purification. 1 Uniblue

A.

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329

Proteins. The following proteins, covering a wide range of molecular weights, were tested: human y-globulin, human transferrin, bovine serum albumin, ovalbumin, a-chymotrypsinogen A, lysozyme, and trypsin (all by Sigma). Surfactants and catalyst. Antarox CO-880 (GAF Corp., Inc., New York) and sodium dodecyl sulfate (SDS; BDH Chemicals, Ltd., Poole, England) were used as commercial grade. The catalyst, 1,4-diazabicyclo-[2,2,2]octane (DABCO) was obtained from Fluka AG, Buchs, Switzerland. Buffer. The pH 10.5 buffer was prepared by mixing 50 ml of 0.05 M sodium bicarbonate with 17.8 ml of 0.1 M sodium hydroxide. Prestaining

Solution.

In a typical prestaining solution containing D, different amounts of protein (lo- 100 pg) from a standard solution, containing 10 mg of protein/IO ml of water, were mixed with 3 mg of dye, 40 mg of urea, 0.2 mg of the catalyst DABCO (16) (in the form of 10 ~1 of 200 mg/lO ml of water), 80 ~1 of buffer, pH 10.5, and were made up to 200 ~1 with water. The mixture was kept at 40°C for 3 hr before electrophoresis. Nonylphenyl polyoxyethylene adduct with 30 ethylene oxide units (Antarox CO-SSO), 0.4 mg, was used instead of urea in the case of trypsin to avoid breaking up the protein into two bands. In the case of lysozyme, Antarox was also used in place of urea, and the reaction was carried out at room temperature for 16 hr so that a sharp protein band was obtained. A pH of 10.5 was chosen for the reaction, because, at this pH, the reaction proceeded at a faster rate without adversely affecting the protein. The reaction could also be carried out at a lower pH. This may be necessary for proteins affected by too alkaline conditions, but, at alower pH, a longer reaction time may be required. Addition of too much DABCO as catalyst can cause tailing of the protein band. With U, DABCO was omitted, because it is not effective; otherwise, the same procedure was used as for D. Electrophoresis Samples of 10, 20, and 50 ~1 of prestaining solution were loaded on an alkaline acrylamide gel (PAG) system as proposed by Ornstein (17) and Davis (18). With y-globulin, transferrin, bovine serum albumin, and ovalbumin, sharper bands were obtained by using 0.1% SDS in the gel and buffer solutions. The samples were electrophoresed at 4 mA/tube until the unreacted dye was clearly separated from the protein band running behind. The gel portion containing unreacted dye was cut off because, in the absence of an electric field, the dye would diffuse throughout the gel. The protein was fixed for 1 hr in a solution containing a mixture of methanol,

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20% (w/v) aqueous sulfosalicylic acid, and 50% (w/v) aqueous trichloroacetic acid in the proportion 5:4: 1. After fixation, all dye not chemically linked to the protein was removed by immersing the gels in 10% (w/v) aqueous acetic acid for several hours. Accurate quantitative measurements were then possible. Quantitation Gel cutting. The cutting instrument used is illustrated in Fig. 1. The gel is placed in a Perspex holder PH- fitted with a cavity according to the gel size used. The cover(C) which is fixed with a pin(P) contains two slits (Sl, S2) 2 mm apart. The Perspex holder with its cover is placed against a locating frame (LF) under a spring-loaded razor blade (B) in such a way that B comes down very quickly into the slit, guillotining the gel suddenly. The depth of the cut can be controlled by an adjustable stopper (ST) so that the razor blade is not damaged by the Perspex holder. In this way a sharp cut and gel slices of almost uniform thickness are obtained. This cutting system can easily be made in any workshop and is much cheaper than the commercially available slicers [e.g., Ref. (19)]. Absorbance Measurements. Absorbance was measured as previously described (7) by placing the gel slice, held in a Delrin plastic holder, in the cell compartment of a spectrophotometer (Hitachi Perkin Elmer Model 139). Measurements were carried out at the appropriate wavelength against an unstained gel slice of the same thickness.

FIG. 1. Gel cutting instrument.

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RESULTS AND CONCLUSIONS

With both dyes, protein bands down to 0.5 pg were clearly visible. Optical densities were measured at 591 (D) and 600 (U) nm over a range of OS-25 pg of protein. The results of a linear regression analysis are given in Table 1. It can be seen that, with the exception of lysozyme, both dyes show about the same sensitivity. However, the scatter, as shown by the correlation coefficients, is always greater with U than with D. The errors in the measurements of any particular amount of protein lie between ?3% and 57% and, sometimes, are higher for optical densities lower than 0. I. Figure 2 illustrates the results obtained with BSA after cutting off the gel containing the unreacted dye in front of the resolving gel. Since one source of error is due to inaccurate loading of the gels with prestained protein solution, a mixture of transferrin and cr-chymotrypsinogen A was prestained with D. If the conversion of protein with dye is the same, the ratio of the absorbances of the two protein bands in one and the same gel should be constant, irrespective of the actual volume of solution applied. The results are summarized in Table 2. As can be seen in Table 2, the ratio of the optical density of stained transferrin to TABLE

1

SLOPESANDCORRELATION COEFFKIENTSOFOPTICALDENSITYICONCENTRATION RELATIONSHIPOF DIFFERENTPROTEINS

Dye

Slope (absorbance/ pg of protein)

Correlation coefficient

Human y-globulin

D U

0.032 0.029

0.9968 0.9954

BSA

D u

0.028 0.036

0.9979 0.9902

Human transferrin

D u

0.022 0.026

0.9985 0.9953

Lysozyme

D U

0.022 0.006

0.9988 0.8256

D U

0.020 0.023

0.9972 0.9965

Ovalbumin

D U

0.019 0.020

0.9994 0.9956

Trypsin

D U

0.017 0.016

0.9981 0.9194

Protein

a-Chymotrypsinogen

A

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FIG. 2. Disc electropherograms on 10% polyacrylamide gel of 0.5-25 c(g of BSA prestained with Drimarene brilliant blue K-BL.

that of stained chymotrypsinogen is constant at different concentrations of these proteins. Because of homogeneous staining right through the gel, as expected, all experiments with D show good linearity of the dye/protein relationship, with correlation coefficients in excess of 0.995. The scatter of up to k7% for a certain amount of protein is not due to any irregularities of the dye-protein reaction, but is chiefly due to the following errors: (a) TABLE RATIO

OF OPTICAL DENSITIES CHYMOTRYSINOGEN

(OD)

OF STAINED

(Ch) AT VARIOUS

Amount of each protein ba)

OJA

O&h

2.50 3.75 5.00 6.25 7.50 8.75 10.00 11.25 12.50

0.057 0.096 0.124 0.143 0.168 0.192 0.222 0.257 0.283

0.047 0.084 0.105 0.123 0.137 0.161 0.192 0.221 0.233

Mean

2 TRANSFERRIN (T) TO STAINED CONCENTRATIONS

ODT/ODch 1.21 1.14 1.18 1.16 1.22 1.19 1.16 1.16 1.21 1.18

Deviation from mean +0.03 -0.04 0.00 -0.02 +0.04 +0.01 -0.02 -0.02 +0.03

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inaccuracies in the preparation of prestaining solutions; (b) inaccuracies in measuring out volumes for loading gels, which could be eliminated by prestaining a mixture of two proteins; (c) slight differences in the thickness of gel slices prepared for absorbance measurements; (d) differences in the smoothness of the surfaces of gel slices. An estimate of amino groups in the protein actually reacted indicated that not all groups had reacted with the dye. With lysozyme and a-chymotrypsinogen A, some of the hydroxyl groups of serine and tyrosine seem to have reacted, which had been noticed previously (8). The stained gels can be exposed to light for a month or more without fading. ACKNOWLEDGMENTS The authors wish to express their thanks to the Australian Research Grants Committee for a grant in aid and to Mr. N. Buchsbaum for his assistance in the experimental work.

REFERENCES 1. Fazekas de St. Groth, S., Webster. R. G., and Datyner, A. (1963)Biochim.Biophys.

Acta

71,377.

2. Fenner, C., Trant, R. R., Mason, D. T.. and Wikman-Coffelt, J. (1975)Anal. Biochem. 63, 595. 3. Reisner, A. H., Nemes, P., and Bucholtz, C. (1975) Anal. Biochem. 64, 509. 4. Kahn, R., and Rubin, R. W. (1975)Anal. Biochem. 67, 347. 5. Bertohni, M. J., Tankersley, D. L., and Schroeder, D. D. (1976) And. Biochem. 71,6. 6. Griffith, I. P. (1972) Anal. Biochem. 46,402. 7. Datyner, A., and Finnimore, E. D. (1973) Anal. Biochem. 52,45. 8. Datyner, A., and Finnimore, E. D. (1973) Anal. Biochem. 55, 479. 9. Talbot, D. N., and Yaphantis, D. A. (1971) Anal. Biochem. 44, 246. 10. Stein, S., Bohlen, P., Stone, J., Dairman, W., and Udenfriend, S. (1973)Arch. Biochem. Biophys. 155,203. 11. Ragland, W. L., Pace, J. L., and Kemper, D. L. (1974) Anal. Biochem. 59, 24. 12. Stein, S., Chang, C. H., Bohlen, P., Imai, K., and Udenfriend, S. (1974)Anal. Biochem. 60,272. 13. Handschin, U. E., and Ritschard, W. J. (1976) Anal. Bjochem. 71, 143. 14. Barger, B. O., White, F. C., Pace, J. L., Kemper, D. L., and Ragland, W. L. (1976)Anal. Biochem. 70, 327. 15. Sun, S. M., and Hall, T. C. (1974) Anal. Biochem. 61, 237. 16. Rys, P., and Zollinger, H. (1975)in The Theory of Coloration ofTextiles (Bird, C. L., and Boston, W. S., eds.), p. 331. Dyers, Company Publication Trust, London. 17. Omstein, L. (1964)Ann. N.Y. Acad. Sci. 121, 321. 18. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. 19. Loening, U. E. (1967) Biochem. J. 102, 251.