Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels

Journal of Biochemical and Biophysical Methods, 20 (1990) 227-235 Elsevier

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JBBM 00801

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels J.R. Vanfleteren i and K. Peeters 2 I Laboratorium voor Morfologie en Systematiek tier Dieren, Rijksuniversiteit Gent, Gent, Belgium and : Department Biochemie, Universitaire Instelling Antwerpen, Wilrijk, Belgium (Received 30 October 1989) (Accepted 27 November 1989)

Summary A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing. Key words: Preparative electrophoresis; Reversed-phase HPLC of protein; Protein recovery; Protein purification

Introduction Frequently the sole applicable tool for separating proteins is sodium dodecyl sulfate gel electrophoresis. While the polypeptide bands can be most appropriately visualized by staining with Coomassie blue, they are also fixed irreversibly within the gel matrix. Stained polypeptides can still be recovered after re-equilibration of the gel in 1-2~ sodium dodecyl sulfate followed by electroelution or electroblotting. However, the yield of the polypeptide material thus eluted is variable and sometimes quite low and upscaling is time-consuming and technically difficult. Correspondence address: J.R. Vanfleteren, Laboratorium voor Morfologie en Systematiek der Dieren, Rijksuniversiteit Gent, Ledeganckstraat 35, B-9000 Gent, Belgium. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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Recently, a rapid procedure has been developed for the visualization of polypeptides separated by sodium dodecyl sulfate gel electrophoresis by simple incubation of the gel in 0.3 M CuCI 2 [1]. The polypeptides are detected as colorless bands against a semiopaque background. The method is fast and claimed to be more sensitive than similar detection methods using KCI [2] or sodium acetate [3] and even more sensitive than Coomassie staining. As the polypeptides are reversibly immobilized within the gel matrix, they will readily elute by simple diffusion after chelation of Cu 2+ with EDTA. In this paper we describe a simple procedure for preparative electrophoresis including sodium dodecyl sulfate gel electrophoresis, copper staining and purification of the eluted polypeptides by reversed-phase HPLC on a fluorocarbon packing material. This approach can be adopted for microscale preparations or it can be upscaled as needed to obtain milligram amounts of (unfolded) protein.

Materials and Methods

Materials Reversed-phase chromatography was carried out using the stainless steel complete HPLC system, LKB (Bromma, Sweden), equipped with a Rheodyne 7125 injector connected to a 1-ml loop. The column was a 2 mm x 10 cm Custom Solvent Miser column (RSL-Alltech Europe, Eke, Belgium) protected by a 2 m m x 2 cm guard colunm (Du Pont Company, Wilmington DE, U.S.A.). The chromatographic support medium used was a polymeric fluorocarbon ('POLY F') HPLC packing material (60 #m particles) from Du Pont. Dry packing material was dispensed separately in bo~a separation and guard columns and settled by tapping the bottom of the column on the benchtop. The support medium was then made wet, and all air removed, by pumping several tens of column volumes of HPLC grade methanol through it at a flow rate of 1 ml/min. Additional packing material was added as needed until both columns were completely filled. HPLC grade water was from Baker (Deventer, The Netherlands), acetonitrile was from Carlo Erba (Milan, Italy). HPLC grade methanol and uvasol grade trifluoroacetic acid were purchased from Merck (Schuchardt, F.R.G.). CuCI2 (research grade) was from U.C.B. (Leuven, Belgium). Bovine serum albumin was obtained from Serva (Heidelberg, F.R.G.), carbonic anhydrase (from bovine erythrocytes), myoglobin (from equine skeletal muscle, type I), cytochrome c (from horse heart) and/~-galactosidase (from E. coli) were from Sigma (St Louis MO, U.S.A.). All c)ther reagents used were analytical grade or of high purity.

Electrophoresis The protein test mixture contained 100 /~g each of /3-galactosidase, serum albumin, carbonic anhydrase, myoglobin and cytochrome c, dissolved in sample buffer (62.5 mM Tris/HCl, 2.3% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 15% glycerol, 0.01% bromophenol blue, pH 6.8). The sample was heated at 90°C for 3 rain and then applied to a 12-cm wide slot of a Tricine gel [4]. The separation gel

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was 14 cm wide, 12 cm high and 0.75 mm thick and contained 105 T (total percentage concentration of acrylamide and bisacrylamide), 35 C (percentage concentration of bisacrylamide to the total concentration T), the stacking gel contained 45 T, 35 C. Gel buffer was Tris/HCl (pH 8.45) in both the separation-(1 M) and stacking-(0.75 M) gel portions. The gels were run in the Bio-Rad (Richmond, CA, U.S.A.) Protean II cell. The upper buffer reservoir contained 0.1 M Tris, 0.1 M Tricine, 0.15 sodium dodecyl sulfate, pH 8.25, the lower reservoir was filled with 0.2 M T r i s / H C I (pH 8.9). Gels were run overnight at 18 mA constant current.

Copper staining Upon completion of the electrophoretic run, the gels were immersed in 500 ml 0.2 M Tris/HCl, 0.2~ sodium dodecyl sulfate, pH 8.8, for 20 rain. Next, they were rinsed with distilled water for a few seconds and immersed in 1 1 0.3 M CuCI 2 for 5-10 min, and kept in double distilled water until further use.

Protein elution Protein containing bands were excised and cut transversaUy into 6-mm wide gel slices, each containing a nominal amount of 5 ltg protein. They were soaked in Milli Q water for 30 rain and transferred into 2-ml screw-capped polypropyle,e tubes (Sarstedt, Herent, Belgium), each tube receiving 1 slice and 1 ml of 0.05 M Na2EDTA/0.05 M Na4EDTA (pH 8.1), 0.1~ sodium dodecyl sulfate was added. The tubes were fixed horizontally on the platform of a gyrotory shaker and shaken at 100 revs/min. Protein was allowed to elute by simple diffusion for 1 day, or longer, at ambient temperature. jS-Galactosidase, which was not amenable to HPLC, was eluted as follows. The copper-stained gel slices were immersed in 0.1 M EDTA (pH 8.1), as just described, and gently shaken for 30 min. Subsequently, they were washed by incubation in four changes (30 rain each) of Milli Q water and finally eluted by diffusion in 1 ml 5 mM Na2HPO4, 0.15 sodium dodecyl sulfate for 1-3 days.

Chromatographic recovery of eluted protein Eluates containing cytochrome c, myoglobin, carbonic anhydrase ~md serum albumin were made AQ 0.2~ F3Ac and loaded directly onto the column. All HPLC separations were performed on the narrow-bore POLY F column eluted with a linear gradient from 0 to 100~ B, followed by 100~ B for 20 rain, where solvent A was 0.11-0.12~ F3Ac (enough to match the optical densities in buffers A and B) and solvent B was 70~ acetonitrile, 0.1~ F3Ac. The flow rate was 200/zl/min. Re-equilibration of the column was with solvent A at a flow rate of 400/zl/min. Absorbance was monitored at 220 nm and 0.64 absorption units full scale and recorded using a chart speed of 5 mm/min. Because of the relatively large tubing (1-ml loop, mixing coil of the instrument) and column volumes relative to the solvent volume consumed during the 20 rain gradient run, proteins seemingly eluted well after the gradient was finished. This effect did not at all compromise the performance.

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Determination of protein recoveries The specific protein recovery rates were determined for the narrow-bore column essentially as described previously [5]. Briefly, samples containing 5 Igg of each test protein (excluding fi-galactosidase) dissolved in 8 M urea, 0.2% F3Ac were chromatographed and the protein peaks were collected in polystyrene microcuvettes. After evaporating the acetonitrile using the Savant SVC200H Speed Vac Concentrator (Savant Instruments, Hicksville, NY, U.S.A.) enough 0.1% F3Ac was added to reach the original volume (approx. 600/tl) and the protein content was determined by the Peterson [6] modification of the Bradford method using appropriate controls, collected from bl~ HPLC runs. The corresponding peak areas in the chromategram were measured using a graphic integrator (Model ID, Summagraphics, Fairfield CT, U.S.A.). The column-specific recovery rates for cytochrome c, myoglobin, carbonic anhydrase and serum albumin were determined by comparing the amounts of protein loaded and recovered as determined by the Coomassi¢ protein assays. The recovery rates of the various test proteins ¢luted from the gel slices and chromategraphed on the POLY F column were then easily quantitated by measuring the corresponding peak areas. Unfortunately, ~-galactosidase was not amenable to HPLC. Its elution rate from the gel slices was then measured in the presence of 0.1~ sodium dodecyl sulfate by the bicinchoninic acid (BCA) protein assay [7] following the Pierce (Rockford, IL, U.S.A.) Micro BCA Protein Assay protocol. Results

Sensitivity of copper staining It was possible to detect 0.1-0.25/~g/lane (7 mm wide) of fl-galactosidase, serum albumin, carbonic anhydrase and myoglobin, which is in good agreement with the sensitivity reported previously [1]. Curiously, bands containing cytochrome c were not readily visible when the protein concentration was below I/~g/lane and even at much higher concentrations cytochrome c stained poorly with copper (Fig. 1). An identical picture was obtained when histone proteins were electrophoresed in sodium dodecyl sulfate gels and stained with copper (results not shown). Thus it may be the intrinsic high positive charge of these proteins that prevents their staining with Cu 2+. HPLC profiles Such sufficiently polar substances as sodium dodecyl sulfate, EDTA and Cu 2+ (whether or not chelated), Tris, Tricine and salts were washed through the column. Variable amounts of uv absorbing material, possibly gel-related components, were retained on the column but usually eluted ahead of the protein peaks (Fig. 2). Protein recovery The recovery rates are summarized in Table 1. The smaller proteins cytochrome c, myoglobin and carbonic anhydrase were completely eluted from the gel slices

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Fig. 1. Preparative separation of a sample containing 100 t~g each of cytochrome c, myoglobin, carbonic anhydrase, bovine serum albumin and p-galactosidase. The sample was loaded into a 12-cm wide slot of a 14× 12×0.75 mm 10~ T, 3~ C sodium dodecyl sulfate Tricine gel. The gel was run overnight at 18 mA constant current and stained with 0.3 M CuCI 2 for 5-10 rain. Cyt c, cytochrome c; Mb, myoglobin; CA, carbonic anhydrase; BSA, bovine serum albumin; /~-Gal, fl-galactosidase. The minor bands possibly represent degraded protein.

within 24 h, as shown by Coomassie staining. In contrast, as shown by the same test, small amounts of serum albumin and fl-galactosidase remained in the gel matrix, even after 3-4 days of elution. It should be emphasized that several bands can be seen on the preparative gel represented in Fig. 1, which represent contaminating or degraded protein. These bands were not excised, of course, and for that reason the recovery rates are actually underestimated. This is particularly the case with carbonic anhydrase (Fig. I and Table 1). Polypeptides recovered by this method were not degraded, as demonstrated by their unchanged mobilities when re-electrophoresed (Fig. 3).

232 B

A ~t

O

m

I I I I I I I I 0 10 20 30 0 10 20 30 min Fig. 2. Elution profiles of myoglobin chromatographed on a fluorocarbon HPLC support. A, a sample containing 5 ttg of myogiobin dissolved in 50/tl 8 M urea, 0.2~ FsAc was loaded onto the column; B, a copper-stained gel slice containing a nominal amount of $ ~tg myoglobin was gently shaken in I ml 0.05 M Na2EDTA/0.05 M Na4EDTA, 0.1~ SDS for 24 h. The eluate was acidified with 0.2~ HsAc and developed on the column. Chromatographic conditions: linear 20 rain gradient from 100~ solvent A (0.11~ aqueous F3Ac) to 100~ solvent B (70~ acetonitrile, 0.1~ F3Ac), followed by 20 rain 100~ solvent B; column: 2 mm × 10 cm, home-packed with a fluorocarbon (POLY F) packing material (60/ira particle size); flow rate: 0.2 ml/min; absorbance was monitored at 220 nm and 0.64 absorption units full scale.

TABLE 1 PROTEIN RECOVERIES (~) FROM COPPER-STAINED GELS a Hours Cytochrome c Myoglobin Carbonic anhydrase Serum albumin /~-galactosidase

24

48

72

96

66 (:I: 5) 64 (:I: 6) 40 (:!: 2) 25 (:!: 3) 24 ( :!:6)

32 36 ( -4-2)

38 39 ( 4- 8)

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a The recovery rates listed for cytochrome c, myoglobin, carbonic anhydrase and serum albumin represent the overall recovery following passive elution and chromatographic purification on a fluorocarbon HPLC chromatographic support medium. Those listed for p-galactosidase represent the elution efficiency only. Average values :i:SE in parentheses are given for triplicate experiments. Cytochrome c, myoglobin and carbonic anhydrase were completely eluted within 24 h, traces of serum albumin and p-galactosidase were still detectable in the gel slices with Coomassie even after 4 days of elution.

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Fig. 3. Sodium dodecyl sulfate gel electrophoresis of proteins recovered from copper-stained gels by passive elution (/|-galactosidase), followed by further chromatographic purification of the eluted protein by reversed-phase HPLC on a narrow-bore (2 mm× 10 cm) colunu~ packed with a fluorocarbon packing material (cytochrome c, myoglobin, carbonic anhydrase and bovine serum albumin). Lane (a), control mixture containing all five test proteins; lane (b), fl-galactosidase; lane (c), bovine serum albumin; lane (d), carbonic anhydrase; lane (e) myoglobin; lane (f), cytochrome c.

Discussion

Procedures for visualizing proteins in sodium dodecyl sulfate gels, yet avoiding their permanent fixation within the gels matrix, have been already presented a decade ago [2,3]. Technical difficulties and lack of sensitivity are the main reasons why these techniques have not been widely employed for recovering proteins by passive elution for further use. The recent copper staining procedure of Lee et al. [1] is much more sensitive (with exception made for very basic proteins), rapid and technically very easy. However, proteins recovered by passive elution are usually present in a high degree of dilution. Such eluates can be concentrated again by steady state stacking [8]. Alternatively proteins can be recovered electrophoretically and collected in a small sample well bounded by a dialysis membrane [9], or they can be concentrated in a conductivity or pH gradieI~t column [10]. Eventually all these methods give rise to protein samples which contain large amounts of low molecular weight substances (buffer salts, urea, SDS, ampholytes) which must be removed prior to amino acid or sequence analysis. An additional dialysis, gel

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filtration or selective precipitation [11,12] step of the recovered protein with organic solvent may result in significant losses, especially when handling low amounts of protein. Sequential application of sodium dodecyl sulfate electrophoresis, copper staining, passive elution and direct purification of the eluate by reversed-phase HPLC on a fluorocarbon packing material appears to be the appropriate approach. The chemical stability of this chromatographic support medium is excellent (pH range 0 to 14). Final recoveries are dependent only on the elution efficiency of the unfolded proteins and their recovery from the column. The recovery rates of the test proteins, spanning a molecular mass range of 11-67 kDa were all nearly or over 90~. As has been generally observed, larger polypeptides amy bind irreversibly to the chromatographic support medium (e.g.//-galactosidase). The very dilute protein samples are concentrated directly on the column, thus avoiding any loss of material associated with classical procedures such as dialysis and lyophilization, and they are released only when the appropriate water/ ace ~onitrile mixture is reached. Because of the high sample capacity of the support medium up to about 0.4 mg of protein will be retained on a 2 mm x 10 cm column packed with 60/~m packing material (measured using serum albumin). Further upscaling can easily be accomplished by using the standard 6.2 mm × 8 cm column (expected capacity at least 3 mg). The need of loading large sample volumes onto the column can be met by a variety of approaches (e.g., multiple injections prior to gradient development; by using some device of 'superloop' (Pharmacia, Sweden); or by aspirating the sample at the low pressure side of pump A (aqueous ~Ac) by means of a three-way valve). The very low back pressure of the POLY F support is an additional advantage (e.g., the back pressure of the present column run at I ml/min of solvent A was only 2-3 bar). Thus large sample volumes can be loaded quickly. Proteins recovered by this method are most likely fully denatured. We are currently using the present procedure to purify a 35 kDa subunit of Daphniapulex (Crustacea) hemoglobin. Preliminary sequence analysis has confirmed that the aminoterminus was still amenable to Edman degradation. The amino acid residues analyzed were not discernably modified by copper staining.

Simplified description and application of the technique A procedure of preparative electrophoresis and chromatographic recovery of the separated polypeptides is described. The protein sample is separated on sodium dodecyl sulfate Tricine or Tris-glycine gels and protein-containing bands are detected by incubating the gels in 0.3 M CuC! 2- The polypeptides are allowed to elute by simple diffusion in 0.1 M sodium EDTA (pH 8.1), 0.1~ sodium dodecyl sulfate and the eluate is subsequently chromatographed on a fluorocarbon (POLY F) HPLC support medium avoiding any other manipulation. Sodium dodecyl sulfate and buffer salts, including EDTA and Cu 2+ are washed through the column, which is then developed in a gradient manner (0-70~ acetonitrile). Unknown uv. absorbing material, possibly gel-related, may elute ahead of the recovered protein. The technique is suitable for micropreparative separations, or it can be scaled up to recover milligram amounts of purified protein., Proteins thus purified are amenable to amino acid sequencing.

235 Acknowledgements This w o r k was s u p p o r t e d b y grant N ° 3.0071.84 f r o m the F u n d for M e d i c a l Scientific Research. J.R.V. is Research D i r e c t o r with the B e l g i u m N a t i o n a l F u n d for Scientific Research. K.P. holds an I W O N L scholarship.

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