ANALYTICAL BIOCHEMISTRY 88, 434-441 (1978)

Polyacrylamide Gel Staining With Fe2+-Bathophenanthroline

Sulfonatel

GARY GRAHAM,~ RODNEY S. NAIRN,~ AND GEORGE W. BATES~

Department of Biochemistry and Biophysics and the Texas Agricultural Experiment Station, Texas A&M University,

College Station, Texas 77843

Received June 6, 1977; accepted March 17, 1978

The utilization of Fez+-bathophenanthroline sulfonate for the detection and quantitation of protein bands in cylindrical polyacrylamide gels is described. Two procedures are outlined. The tirst procedure is used in standard disc electro- phoresis and involves fixing the protein with trichloroacetic acid, staining with Fez+-bathophenanthroline sulfonate, and destaining with an ethanol:acetic acid solution. The second protocol reported is utilized with sodium dodecyl sulfate- containing gels. After electrophoresis, the gels are incubated with a methanol: acetic acid solution to remove the sodium dodecyl sulfate. The gels are then stained with Fe*+-bathophenanthroline sulfonate and destained with a methanol: acetic acid solution. Excellent background clarity is observed with both methods. Densitometric areas of the stained protein bands are linear to 60 pg of bovine serum albumin, and the limit of detection of this protein is 1 pg. Because of its rapidity of staining and destaining, good sensitivity, and reproducibility of stain intensity, Fez+-bathophenanthroline sulfonate is an excellent protein stain.

Polyacrylamide gel electrophoresis is a widely used technique for the separation of proteins. The salient characteristics of the technique have been described (1,2). Various procedures for the visualization of proteins after separation have been reported. In recent years, the introduction of acid wool dyes such as Coomassie blue and amido black have greatly improved the sensitivity of these techniques (3-6). Coomassie blue has been reported to be the most sensitive of this type of stain and has been utilized to identify protein in microgram quantities (6). Unfortunately, quantitative methods using these dyes require lengthy staining and de- staining times and are linear over a narrow concentration range (3,5,6). This paper describes the use of Fe 2+-bathophenanthroline sulfonate

’ This work was supported by Research Grant A-430 of the Robert A. Welch Foundation, Houston, Texas.

’ Texas A&M University Health Fellows. B To whom inquiries should be addressed.

0003-2697/78/0882-0434$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

434

Fe*+-BATHOPHENANTHROLINE SULFONATE GEL STAINING 435

(Fez+-BPS) to detect and quantitate proteins on cylindrical polyacryl- amide gels. The structure of BPS is shown in Fig. 1. This ligand forms a 3:l complex with iron which has a molar absorptivity of 2.21 x lo4 liters mol-’ cm-l at 533 nm. The techniques reported are recommended for their rapidity, high sensitivity, and reproducibility of stain intensity. Fez+-BPS has proven useful in both standard disc electrophoresis and SDS electro- phoresis.

MATERIALS AND METHODS

Reagents. Bovine serum albumin (BSA) monomer was purchased from Miles Laboratories, Inc. (Elkhart, Ind.). Lyophilized human serum was purchased from Hyland Laboratories (Costa Mesa, Calif.). Sodium batho- phenanthroline sulfonate (BPS) was obtained from Sigma Chemical Co. (St. Louis, MO.). Sodium dodecyl sulfate (SDS) was purchased from Pierce Chemicals (Rockford, Ill.). Acrylamide, N,N’-methylenebisacrylamide, and N,N,N’,N’-tetramethylethylenediamine (Temed) were obtained from Bio-Rad Laboratories (Richmond, Calif.). All other chemicals were of reagent grade or better and not further purified.

The staining solution for standard disc electrophoresis contains: Fez+, 1 mM; BPS, 3 mrvr; ethanol, 40% (v/v); and acetic acid, 5% (v/v). The active staining material is the Fe *+-BPS complex, and the protein bands are stained bright pink. To prepare 200 ml of this solution weigh out 0.078 g of Fe(NH,),(SO,),.6H,O. Dissolve this in approximately 15 ml of 10% acetic acid. To this solution add 20 ml of 30 mM BPS and allow the mix- ture to react for 5 min. Next, add 80 ml of ethanol and then dilute to volume with 85 ml of 10% acetic acid. The staining solution used with SDS gels is prepared with methanol instead of ethanol.

Electrophoresis. Clarke’s (7) modification of the techniques described by Davis (2) was used in most of our electrophoretic separations. For electrophoresis of serum samples a stacking gel was employed. Separating gels (7.5%, w/v) were formed in Photo-F10 (Eastman Kodak)-coated cylin- drical glass tubes (5 mm in diameter, 12.5 cm in length). Polymerizing gels were overlayed with water-saturated isobutanol. Large-pore stacking gels used for serum samples contained 3.4% (w/v) acrylamide and were chem- ically polymerized.

SDS-Containing gels were cast using the acrylamide to bisacrylamide ratio described by Lugtenberg et al. (8). The separating gels contained:

/\ \-I - -

5l-Q-Y , \ , \ -2S03Na

-N N-

FIG. 1. The structure of bathophenanthroline sulfonate (BPS). This chromogenic chelating agent forms a 3: 1 chelate:Fe2+ complex.

436 GRAHAM, NAIRN, AND BATES

Tris-HC10.375 M, pH 8.8; acrylamide, 8.8% (w/v); SDS, 0.2% (w/v); and EDTA, 0.002 M. The gels were chemically polymerized with ammonium per-sulfate (0.025%, w/v) and Temed (0.25%, v/v). Chemically polymerized large-pore SDS gels contained 3.4% (w/v) a&amide prepared from a stock solution containing 22.5 g of acrylamide and 0.6 g of bisacrylamide per 100 ml.

Electrophoresis was carried out using a water-cooled Bio-Rad Model 150A gel electrophoresis cell (Bio-Rad Laboratories, Richmond, Calif.) and an LKB Model 2103 power supply (LKB Instruments, Rockville, Md.). All experiments were run at a constant power of 0.88-1.0 W/tube. The electrode buffer for standard gels was that of Davis (2). For SDS gels the electrode buffer was 0.19 M glycine, 25 mM Tris, 2 mM EDTA, and 0.1% (w/v) SDS.

Staining. The gels (except SDS) were fixed in 10% (w/v) trichloroacetic acid (TCA) for 1 hr at room temperature. After being washed with dis- tilled water, the fixed gels were individually stained in test tubes using the ethanol-containing stain for 20 min at 65°C. The staining solution was de- canted and saved, and the unwashed gels were individually destained for 50 min at 65°C using an ethanol:acetic acid:water (5:2: 13) destaining solu- tion. All fixing, staining, and washing steps were carried out in a total vol- ume of 25 ml. This resulted in a solution:gel ratio of 1O:l. At this point in the procedure a qualitative identification of the protein bands can be made. For a quantitative estimation of the amount of protein present, the gels were transferred without washing to water and kept at 65°C for 4 hr. The resultant gels have completely clear backgrounds. Subsequent storage of stained gels in water resulted in no demonstrable loss of intensity of stained bands. Densitometrically, this result was observed up to about a week of storage in water. Visually, there is no noticeable decrease in in- tensity of stained bands up to several months of storage.

SDS gels were fixed overnight in an aqueous solution of 50% (v/v) methanol and 10% (v/v) acetic acid at 65°C. The gels were then thoroughly washed with water and allowed to stand in water for 1 hr at 65°C before staining. Next, the gels were stained with the methanol-containing Fez+- BPS solution for 20 min at 65°C. Destaining was performed at 65°C with the 50% (v/v) methanol and 10% acetic acid solution for 5 hr. The destain- ing solution was changed twice during this period. The partially destained gels were then washed with water and immersed in water at 65°C for 90 min. The resultant gels have a completely clear background. All gels were stored in water at room temperature. Sodium azide (0.02%, w/v) was added when gels were stored for long periods.

Densitometry and photography. A Photovolt Model 542 Densicord den- sitometer with a cylindrical gel-scanning apparatus was used to quantitate the stained protein bands (6). A 545-nm filter was used in quantitating bands stained with Fez+-BPS. For comparison some gels were stained with a

Fe*+-BATHOPHENANTHROLINE SULFONATE GEL STAINING 437

relatively fast Coomassie blue staining method described by Malik and Berrie (9). These gels were scanned using a 610-nm filter. The areas of the peaks were measured by cutting out the tracing of the peak and weighing it on an analytical balance. The area of the peak was then calculated from the weight of the tracing of the peak and the weight of a piece of paper of known area.

Photographs of the stained gels were obtained using a Polaroid MP-3 camera system and Polaroid Type 55 positive-negative film. Illumination was provided by a light box. A Wratten No. 23A filter was used to photo- graph gels stained with Coomassie blue and a Wratten No. 65A filter was used for the Fez+-BPS-stained gels. All photographs were taken with water-filled overlay tubes (10).

RESULTS AND DISCUSSION

Staining Purameters

Initial experiments using Fe *+-BPS were aimed at determining the optimal conditions for staining and destaining. Experiments were run in which series of tubes each containing 10 pg of BSA were electrophoresed and stained for times varying between 5 min and 2 hr. Twenty minutes was chosen as an optimal staining time since no quantitative increase in band intensity was found after this time.

Prolonged staining was found to increase markedly the time required in the destaining step in order to achieve an acceptable background clarity. The temperature used in many staining procedures, 65°C) was found to be quite suitable for staining with Fe?+-BPS.

The optimal procedure for destaining gels is presented above. We found that the bands intensify in color during the destaining process, whereas the background clears. For this reason it is important that the exact pro- cedures described above are followed. It is important to note that the destaining process has two stages. The first stage requires heating in ethanol:acetic acid, and the second, heating in water. Intensification of the bands appears to occur during both stages. On occasion, when satis- factory background clarification was not obtained, the water in the tubes was poured out and replaced with fresh water, and the gels were incubated for an additional 30 min. This brought about highly satisfactory back- grounds in these cases. When carried out as described, the Fez+-BPS stain allows one to visualize bands conaining 1 to 2 pg of BSA.

Quantitation of Protein Content of Bands

Figure 2 shows a series of gels in which the amount of BSA, the major band, varies from 2.5 to 60 pg. It is apparent that visualization of the 2.5-pg sample is quite satisfactory. We compared our rapid Fez+-BPS

438 GRAHAM, NAIRN, AND BATES

FIG. 2. A series of acrylamide gels containing BSA which were stained with Fez+-BPS. The amounts of BSA present were, from left to right, 2.5, 5, 10, 20, 30, 40, 50, and 60 pg. Staining and destaining were performed as described under Materials and Methods. The pro- tein was applied in a lo-p1 volume and was weighted with 7% sucrose.

staining method with the rapid Coomassie blue method of Malik and Berrie (9). This method is the most rapid Coomassie blue technique de- scribed in the literature. In these experiments, we modified the method of Malik and Berrie by staining for 4 hr at 65°C rather than the reported 30 min at room temperature. This led to significant intensification of the Coomassie blue-stained protein bands. Despite this modification, the Fez+- BPS stain proved definitely superior in visualizing bands in the 0.6- to 5-pg range. It is this level of protein content that is important in efforts to extend the utility of rapid staining techniques. We also compared our rapid method with a slow but sensitive Coomassie blue technique de- scribed by Fishbein (6). While this technique definitely allows better vis- ualization of protein bands containing 0.2 to 0.6 pg, Fishbein’s procedure requires 3 days, whereas ours requires approximately 6 hr. The superior clarity of Fe*+-BPS-stained gel backgrounds is advantageous in den- sitometric scanning of the gels.

Figure 3 shows the linear response obtained from densitometric scans as a function of the protein content. The response is linear to approxi-

Fez+-BATHOPHENANTHROLINE SULFONATE GEL STAINING 439

PROTEIN (Fg)

FIG. 3. A plot of densitometric area versus amount of BSA (in kg) applied. The protein was applied in a 10-w] volume and was weighted with 7% sucrose. Response setting of the densitometer was “F”.

mately 60 pg after which there is a sharp break, characteristic of den- sitometric quantitation of dye-binding techniques (6). The closeness of fit of the points to the curve is an indication of an acceptable precision.

A drawback to some methods which are sensitive to microgram amounts of protein is inferior reproducibility. In a series of six independent experi- ments using identical concentrations of BSA, areas under the curves ob- tained from the densitometer tracings were determined and found to be essentially identical. The reproducibility of the staining technique is at least as precise as that of the densitometer.

Comparison of Fez+-BPS with a Rapid Coomassie Blue Method Using Human Serum

Figure 4 depicts the results of an experiment in which human serum was subjected to standard polyacrylamide gel electrophoresis and subsequently stained with the Fe2+-BPS method (A), or our modification of the rapid Coomassie blue method of Malik and Berrie (B). Accompanying the gels are the corresponding densitometer tracings. A candidate for staining of protein bands should not exhibit high selectivity with regard to proteins, but should stain all proteins equally. In Fig. 4 it is evident that all of the bands stained with Coomassie blue also stained with the Fe2+-BPS. It is possible to discern additional bands in Fig. 4A. Several bands show a greater intensity as compared with those stained with Coomassie blue. The densitometer scans quantitate the enhanced intensity observed with Fe*+-BPS staining.

440 GRAHAM, NAIRN, AND BATES

$JJy-JJ 6 7 8

MIGRATION DISTANCE(cm)

A

tJ--Q-J

0 I 2 3 4 5 6 7 8 MIGRATION DISTANCE(cm)

B

FIG. 4. A comparison of Fe*+-BPS (A) and Coomassie brilliant blue (B) staining of human serum. The serum sample was diluted 1: 1 with a solution containing 15% sucrose and 0.5% bromophenol blue. A 5Oy.d aliquot of the diluted sample was applied to each gel. Elec- trophoresis was performed for 2.5 hr at 1 W/tube.

Staining of Gels Containing SDS

SDS electrophoresis is a widely used technique that has a range of ap- plications. Unfortunately, this technique is limited somewhat by the time required for staining and destaining of the gels. For example, commonly used Coomassie blue techniques require 48 hr or longer when applied to SDS-containing gels or must employ electrophoretic destaining. We have found that Fez+-BPS does not bind to gels containing SDS and that it is necessary to fix the protein bands and wash away the SDS before staining. The details of the SDS removal procedure in the methanol:acetic acid solvent are described above. We have found it convenient to incubate the gels at 65°C overnight. This allows sufficient removal of the SDS so that subsequent washing and a heating in water will allow the protein to be

Fe*+-BATHOPHENANTHROLINE SULFONATE GEL STAINING 441

stained. We should emphasize that incomplete removal of SDS prior to staining causes a lower intensity of staining.

The observation that Fe?+-BPS does not bind to proteins in the presence of SDS, an anionic detergent, suggested to us that the binding may be of a noncovalent nature. To test this we used Teepol 610 (Shell Oil Co.). another anionic detergent, which has been used to test for covalency of attachment of a dye ( I 1). We found a complete bleaching of the bands after 24 hr, indicating that Fe?+- BPS is attached in a noncovalent fashion to these protein molecules.

ACKNOWLEDGMENTS

The authors express their appreciation for the technical assistance and advice of C. R. Young. C. W. Dill. and D. L. Zink.

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Acta 71, 377-391. 4. Meyers, T. S.. and Lamberts, B. L. (1965) B&him. Biophys. Acru 107, 144-152. 5. Chrambach. A.. Reisfeld, R. A., Wyckoff. M., and Zaccari, J. (1967) Anal. Biochem.

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