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Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels
ANALYTICAL
BIOCHEMISTRY
171,352-359
(1988)
Isolation of Proteins for Peptide Mapping, Amino Acid Analyses, and Sequencing Using Disulfide Crosslinked Polyacrylamide Gels’ SEYED H. GHAFFARI,
TAMBA S. DUMBAR, MICHAEL AND MARK 0. J. OLSON~
0. WALLACE,
Department of Biochemistry, The University OfMississippi Medical Center, 2500 North State Street, Jackson, Mississippi 392164505 Received August 28, 1987 Conditions for recovery of small amounts of proteins (l-50 pg) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilixation and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and &galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments. Q 1988 AC&I& yes, IOC. KEY WORDS: sodium dodecyl sulfate-polyacryamide gel electrophoresis; protein precipitation; amino acid analysis; protein sequencing.
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDSPAGE)3 is a powerful tool in protein biochemistry because of its ability to resolve complex mixtures of polypeptides present at very low concentrations. Recently, methods have been developed for recovery of small quantities of protein from gels for peptide mapping (l), amino acid analyses (2), or protein sequencing (3-5). These techniques involve either electrophoretic elution from gel slices or electrophoretic transfer to nitrocellulose or glass fiber membranes. Although these are generally effective methods for ob’ This work was supported by Grants GM28349, RR 02745, and RR 05386 to M.O.J.O. from the National Institutes of Health. 2 To whom correspondence should be addressed. 3 Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BAC, N,N’bisacrylylcystamine; Bis, N,N’-methylenebisacrylamide; NCS, N-chlorosuccinimide; PTH, phenylthiohydantoin; TCA, trichloroacetic acid; UHA, urea:water:acetic acid, TEMED, N,N,N,N’-tetramethylethylenediamine. 0003-2697188 $3.00 CopMgbt Q 1988 by Academic Pms, Inc. AI1 rights of reproduction in any form -cd.
taming small quantities of certain proteins, there are a number of difficulties associated with their use: (i) they are cumbersome, time consuming, and require multiple sample manipulations; (ii) they expose the sample to large volumes of solvent, often resulting in contamination; (iii) they require special apparatus; (iv) they often result in low yields of recovery; and (v) they require special pretreatment of the glass fibers onto which the polypeptides are blotted for sequence analyses. Because of these difficulties we have explored an alternative method for isolation of microgram quantities of proteins. A variety of reversible crosslinkers for polyacrylamide gels have been available since the mid- 1970s. These include N,N’-diallyltartardiimide (6), N,N’-dihydroxyethylenebisacrylamide (7), and N,N’-bisacrylylcystamine (8). We have focused on the latter reagent because it is possible to dissolve the gel using relatively mild, neutral, and nonoxidizing conditions. Despite these favorable properties only a few 352
DISULFIDE
GEL
PROTEIN
reports have utilized the disulfide gels for protein isolation. The purpose of this study was to develop methods utilizing the disulfide gel system for isolating microgram or nanogram quantities of protein suitable for use in peptide mapping, amino acid analyses, or protein sequencing. In this paper we describe methods which may be used for all three purposes. MATERIALS
AN5
METHODS
Chemicals and proteins. Most developmental experiments utilized the high-molecular-weight standard mixture from the Sigma Chemical Co. which contained the following proteins (and respective molecular weights): bovine carbonic anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000), rabbit phosphorylase b (97,400), Escherichia coli P-galactosidase (116,000), and rabbit myosin (205,000). In some experiments the molecular weight standard mixture was labeled with ‘25I using the chloramine-T procedure (9). Sperm whale myoglobin (sequencer grade) used in the sequencing studies was obtained from Beckman Instruments. Polyacrylamide, N,N’-methylenebisacrylamide (Bis), N,N’-bisacrylylcystamine (BAC), and N, N, N,N’-tetramethylethylenediamine (TEMED) were purchased from Bio-Rad. Polybrene was purchased from Applied Biosystems, Inc. Partially purified nucleolar protein fractions were obtained from Novikoff hepatoma nucleoli as previously described ( 10). Electrophoresis procedures. Proteins were run on a gel system essentially as described by Flyer and Tevethia (11) using the reversible crosslinker BAC. A 20% stock solution was made with a ratio of acrylamide to BAC of 19.22:0.78 (w/w). The separating gels containing either 7.5 or 10% acrylamide/BAC were made in TBES buffer (to final concentrations of 89 mM Tris-HCl, 89 mM boric acid, 2.8 mM EDTA, 0.1% SDS, pH 8.3) and were thoroughly degassed before polymerization (see Table 1 for composition of gels).
ISOLATION
353
Most gels were cast as 140 X 160 X l-mm slabs in the Bio-Rad Protean I apparatus, although some runs were made in the Mini Protean II system (60 X 80 X 0.75 mm). After pouring, the gels were overlayered with isobutanol. The separating gels were preelectrophoresed in TBES buffer for 3-4 h at 100 V to remove excess reagents and unreacted polymerization products and to equilibrate gels to the proper pH and ionic strength. Due to the presence of mercaptoethanol in sample buffer, stacking gels were of the standard Laemmli type (I 2) containing 5% acrylamide and 0.13% Bis. Samples were applied to the gels with Laemmli-type sample buffer. For evaluation of purity of isolated proteins and peptide mapping, standard Laemmlitype gels were used. After electrophoresis the gels were stained for 1-2 h in 0.1% Coomassie blue in 50% methanol containing 10% acetic acid and destained in a solution containing 10% isopropanol and 10% acetic acid. Isolation of proteins ,from gels. Stained bands were excised, washed for 1 h in water with four changes, cut into small pieces, and transferred to microcentrifuge tubes. Two different procedures for dissolving the gel were used. In the first, l-2 vol of 2-mercaptoethanol plus 1 vol of water (to lower the viscosity) were added. In most cases the gels dissolved within a few minutes. In some instances where the gels did not readily dissolve, the tubes were heated at 80-90°C for 5 min and gently shaken until the gel was liquefied (usually 5-30 min). In the second method, 2 vol of 1 M dithiothreitol in 0.05 M sodium phosphate buffer (pH 7.0) was added to the gel slices, which were then incubated at 70°C for 15 min. Concentrated trichloroacetic acid (TCA) (40%) was added to a final concentration of 15%. In some peptide mapping experiments, polyadenylic acid (poly(A)) carrier was added at a concentration of 40 pg/ml. Also, for some samples subjected to amino acid analyses or sequencing, Polybrene (40 pg/ml) was added prior to the addition of TCA. The tubes were allowed to
354
GHAFFARI
stand at 0-4°C overnight and then centrifuged at 10,OOOg for 30 min in a microcentrifuge at 4°C. The supernatant was decanted and the pellet was washed once as above with 15% TCA. To remove the TCA the pellet was washed twice with ethanol:ether (1: 1, v/v) and once with ether (13). Peptide mapping. In most experiments chemical cleavage was performed before the protein was removed from the gel. Slices containing stained protein bands were washed with 25 ml of water two times for 10 min. For N-chlorosuccinimide (NCS) cleavage the gel slices were subsequently washed two times for 10 min with 10 ml of urea:water:acetic acid ( 1 g: 1 ml: 1 ml; UHA). Then 5 ml of 0.015 M NCS in UHA was added and the cleavage reaction (14) was continued for 1 h at room temperature. For CNBr cleavage the gel slices were washed with water and then with 4 ml of 70% formic acid for 30 min at 25°C. The slices were then incubated with 2 ml of 1% CNBr in 70% formic acid for 1 h at room temperature in the dark. Immediately after the cleavage reactions the slices were washed with water as above, followed by a 20-min wash in running buffer (25 ml) and a 20-min wash in 4 ml of sample buffer which did not contain mercaptoethanol. The slices were then dissolved by adding 20 ~1 (per slice) of sample buffer containing l-5 ~1 of 2-mercaptoethanol. The dissolved slices were heated at 100°C for 3 min and then applied directly to Laemmlitype gels. Other peptide mapping experiments used proteins which had been removed from the gels and precipitated. In these, the cleavage reactions were carried out essentially as above except that reaction volumes were reduced to 50 ~1. After cleavage the samples were diluted with water ( 10 vol) and freezedried before application to the gels. Amino acid analyses and sequencing. The preferred procedure for sample handling prior to hydrolysis was to perform all manipulations in the hydrolysis tubes. With samples for which this could not be done, precip-
ET
AL.
itated proteins were first dissolved in 50 ~1 of 5.7 N HCl (Pierce Sequenal grade) by heating for 5 min at 100°C and transferred to 6 X 50-mm tubes. The samples were dried in a Savant Speed-Vat and subjected to hydrolysis for 22 h at 110°C in a Waters Picotag work station. Amino acid analyses were performed on a Beckman 119CL amino acid analyzer. Protein sequencing was done on an Applied Biosystems 470A sequencer using the 03RPTH program. The precipitated protein samples were dissolved in 30-60 ~1 of l-4% SDS or 70% formic acid and applied to the Polybrene-treated glass fiber filter. PTH amino acids were identified on a Waters HPLC system which was directly interfaced with the sequencer via a Rheodyne 7126 injection valve. In this system approximately 40% of each residue was applied to an Applied Biosystems dedicated PTH amino acid column (2.1 mm) using the solvent system specified by the vendor. RESULTS
Choice of gel electrophoresis conditions, Because of the high resolving power of Laemmli-type gels we attempted a simple substitution of the BAC crosslinker for Bis in initial experiments. This was generally unsuccessful because of difficulties in obtaining the correct level of crosslinking and the inability of certain proteins to enter the gel. Therefore, we adopted the system used by Flyer and Tevethia (1 l), using gels containing either 7.5 or 10% polyacrylamide (Table I). Resolution on this system was adequate for either standard protein mixtures (Fig. 1) or samples of partially purified cell fractions. Furthermore, the use of the Tris-borate buffer system rather than Tris-glycine helped to reduce glycine contamination for amino acid analyses and sequencing (see below). Recovery of proteins from gels. In TCA precipitation of proteins it is a common practice to add a carrier to improve recovery
DISULFIDE TABLE
GEL PROTEIN
1
COMPOSITIONOFPOLYACRYLAMIDESEPARATINGGEL SOLUTIONS WITH BACCROSSLINKER
% Acrylamide 7.5
10
ml of component per 40 ml gel 20% acrylamide/BAC stock” 10x TBES* Hz0 TEMED 10% (NH.,)&Os
15 4 20 1
20 4 14.8 1.2 0.06
0.06
’ Made with a ratio of acrylamide:BAC of 19.22:0.78 (w/w). * 1X TBES contains 89 mM Tris-HCI, 89 mM boric acid, 2.8 mM EDTA, and 0.1% SDS, pH 8.3.
of small amounts of protein. For this purpose we have routinely added poly(A) to the dissolved gel slices prior to TCA precipitation. This has been satisfactory for mapping stud-
ies, but the poly(A) was a source of contamination for amino acid analyses and protein sequencing. It was discovered that reasonable recoveries were possible in the absence of poly(A) or other carriers. The recovered proteins could then be resolubilized in l-4% SDS for further analyses. By gel electrophoresis the recovered proteins had migration characteristics essentially identical to those of the original material and were of high purity (Fig. l), thereby providing the initial evidence for the usefulness of the method. To determine the efficiency of recovery of protein by the TCA precipitation procedure, radioiodinated protein samples were run at various concentrations on the gel system. The stained bands were excised. counted in a gamma counter, dissolved, and precipitated, and the precipitates were counted again. Table 2 indicates that the recovery varied from about 30 to nearly 90%, although most samples were recovered in the range 60-70%. The recovery was dependent on the protein as well as on the concentration of the protein
II EA
BA
1301, C23
Ill
I
B
*
355
ISOLATION
4160K 4C23
,~%* 1 4 160 K 4 130 K 4 C23
FIG. I. Isolation of proteins from disulfide gels. (A) The commercially available high-molecular-weight mixture of proteins (Sigma) was applied to a 10% polyacrylamide gel using N,N’-bisacrylylcystamine (lane I). After electrophoresis the stained bands of bovine serum albumin (BA) and ovalbumin (EA) were cut out, dissolved with mercaptoethanol, precipitated with 10% TCA, and washed with ethanol/ether. The lanes in II show the reelectrophoresis of the precipitated proteins using 10% Laemmli-type gels. (B) Partially purified nucleolar extracts (I and II) were applied to 10% disulfide gels as in A. After electrophoresis the stained bands as indicated were applied to the second gel as in A.
356
GHAFFARI
ET AL.
TABLE 2 RECOVERIESOFPROTEINSFROMSTAINEDBANDS Percentage recovered Micrograms loaded” 50
20 10 5 1
Ovalbumin
Phosphorylase b
/3-Galactosidase
73.2 76.2 61.4 32.5 23.4
62.7 65.7 61.7 68.6 86. I
58.8 75.1 40.2 65.6 56.5
” The radioiodinated proteins were applied to 10% disulfide gels and isolated by TCA precipitation without carrier. The recoveries were calculated from the cpm in each band before the gel was dissolved and the cpm recovered in the pellets after precipitation. Each value is based on duplicate determinations. The range for the duplicates was as much as 10%.
precipitated. For example, the percentage recovery of albumin was considerably lower at low concentrations than at high concentrations. On the other hand, the recovery of phosphorylase b was generally high (about 65%) and less dependent on the concentration of protein in the gel band. It was also found that reasonable recoveries could be expected down to 1 pg of protein. However, recovery of low-molecular-weight proteins was very low; e.g., insulin A or B chains (M, 2300-3400) were recovered in yields of less than 10% (data not shown). On the other hand, apo-myoglobin (Mr 18,360) was recovered in sufficient quantities for sequencing (see below). Thus, the lower limit for effective precipitation would appear to be somewhere between A4,3400 and M, 18,000. Peptide mapping of recovered proteins. Peptide mapping was done with CNBr and NCS on test proteins purified on the BAC gel system. The most convenient procedure was to perform the cleavage reaction in the gel slice before the gel was dissolved. The dissolved slices could then be applied directly to the second gel to yield clearly defined banding patterns (Fig. 2). The advantage of this procedure over mapping methods (14) which employ conventional Bis crosslinked gels is that multiple bands of purified protein can be applied to the same lane of a gel. In ex-
periments not shown when more than two tional gels were applied gel, smearing resulted the cleavage products
CNBr EA BA
it was observed that bands from convento a lane of a second presumably because were removed from
WCS El
BA
FIG. 2. Peptide mapping of proteins isolated from disulfide gels. The commercially available high-molecular-weight mixture of proteins (Sigma) was applied to a 10% polyacrylamide gel using N,N’-bisacrylylcystamine as in Fig. IA. After electrophoresis the stained bands (designated by small letters) were excised, subjected to cleavage with either cyanogen bromide (CNBr) or Nchlorosuccinimdie (NCS), and solubilized as described under Materials and Methods. The cleaved proteins were reelectrophoresed on 12% Laemmh-type gels.
DISULFIDE
GEL
PROTEIN
the first gel slices at variable rates. Dissolving the gel allowed the fragments to form a homogeneous band at the top of the gel prior to separation. In some experiments in which the protein in the stained bands was very dilute and the volume which could be accommodated by the sample slots was limiting, cleavage was done after precipitation. In these (not shown) the cleavage pattern was essentially indistinguishable from the results shown in Fig. 2, although this method was somewhat less convenient. Amino acid analyses of recovered proteins. Several standard proteins were isolated from gel bands by reversing the crosslinking and TCA precipitation. These were subjected to hydrolyses and conventional amino acid analyses. For comparison, samples of the purified proteins obtained commercially were subjected to hydrolyses and amino acid analyses under identical conditions but without further purification. Table 3 indicates that for all proteins analyzed the compositions of the gel-purified proteins were remarkably similar to those of the commercially available preparations. It is not possible to determine whether the minor differences are due to a higher purity of the gel-purified proteins or to minor contaminants introduced by the method. In any event, these results indicated that reliable amino acid compositions could be obtained from gel-purified proteins. Protein sequencing of disuljide gel-purified protein. The final test of the disulfide gel purification method was to perform protein sequencing on a standard protein. For this purpose, whale myoglobin (approximately 20 pg) was applied to the gel system, purified, and precipitated without carrier. Heating the precipitated sample at 100°C for 3 min in 1% SDS solubilized about 70% of the protein as measured by amino acid analysis. We also found 70% formic acid to be an effective solvent, giving nearly complete recovery without heating the sample. An aliquot of the solubilized protein was applied to a precycled glass fiber filter of the gas phase sequencer. The results of 12 cycles of Edman
357
ISOLATION
degradation were comparable to results obtained from commercially purified standards; the initial yield was 9 1% and the repetitive yield was 93.5%. This is well within the range specified by the manufacturer for the test protein; the repetitive yield should be at least 92% and 94% for 100 pmol and 1 nmol of myoglobin, respectively. Although the runs were not carried beyond cycle 13, the repetitive yield suggests that extensive Edman degradations could be carried out on this material. Furthermore, the level of contaminants was no higher than that in runs made with conventionally purified proteins, although the glycine and proline background was 2-4 pmol higher than normal. Thus, the method appears to be useful for purification of small amounts of proteins or peptide for amino-terminal sequence analyses. We have successfully applied this technique to several polypeptides of known and unknown sequence, e.g., P-galactosidase and CNBr fragments from nucleolar protein C23. For sequencing we have found that addition of Polybrene (1) as a carrier aids in recovery and transfer of the protein to the filter disk of the sequencer. DISCUSSION
The data presented in this paper indicate that by altering the crosslinking agent a polyacrylamide gel electrophoresis system can be used to purify proteins for peptide mapping, amino acid analyses, and sequencing. This method, which provides an alternative to electrotransfer and electroelution methods, utilizes ordinary electrophoretic and laboratory equipment. A possible disadvantage is that the resolving power of the gel system used in these experiments is slightly less than that of Laemmli-type systems. Using the 7.5 and 10% acrylamide systems discussed in this paper, resolution was adequate for polypeptides between M, 40,000 and A4,200,000; below M, 40,000 the band spreading would require a prefractionation step to make the starting material less complicated. The reso-
358
GHAFFARI
lution may also be decreased by interaction of sulthydryl groups in the protein with the gel matrix, although we have not systematically examined this. Thus, for highly complex protein mixtures, reduction and carboxymethylation may be required. In the attempts to adapt the disulfide gel system to the three techniques discussed, some difficulties were encountered. First, it was necessary to adjust the concentrations of ammonium persulfate and TEMED to control the rate of polymerization. The amounts indicated in Table 1 were determined using the guidelines outlined in Bio-Rad Technical Bulletin 2045. These concentrations have been satisfactory for several batches of BioRad chemicals, but adjustments may be necessary for other batches or for chemicals from other vendors.
ET AL.
The second problem was the tendency of protein to stick at the interface between the stacking and the separating gel. This was solved by preelectrophoresis of the gel immediately after polymerization to remove the reactants and charged reaction products. The final problem was that of solubilizing the gel containing the stained protein band. Solving the first two problems largely solved this problem by preventing formation of covalent crosslinks. However, it was also necessary to maintain an adequate ratio of the volume of mercaptoethanol to the volume of the gel slice. At least a 2: 1 ratio was required for the gel slices to dissolve and for the viscosity of the solution to be low enough for TCA precipitation to take place. We also found that 1 M dithiothreitol was a much more effective solubilizing agent than mer-
TABLE 3 AMINO
ACID
COMPOSITIONS
OF GEL-PURIFIED
AND
CONTROL
PROTEINS
mol % Amino acid“ Asx Thr Ser GIX
Pro GlY Ala Val Met Be Leu TY~ Phe His LYS A%
Bovine albuminh 9.0 (9.5) 5.1 (5.4) 4.6 (5.7) 16.8 (14.1) 5.8 (7.1) 4.1 (2.8) 7.7 (7.9) 5.4 (7.2) 0.9 (0.6) 2.8 (0.6) 17.2 (15.7) 2.0 (2.8) 4.3 (4.9) 2.3 (2.3) 7.9 (10.2) 4.2 (4.4)
Ovalbumin 9.7 (10.1) 3.7 (4.2) 9.0 (9.3) ll.9(12.1) 5.7 (4.3) 7.7 (6.2) 8.6 (9.4) 8.1 (8.9) 2.3 (3.2) 4.7 (4.8) 9.1 (8.6) 1.9 (1.9) 5.2 (5.0) 2.1 (1.6) 5.3 (5.8) 4.9 (4.8)
Phosphorylase b
P-Galactosidase
11.3 (11.7) 4.5 (4.2) 4.3 (2.9) 12.5 (12.5) 5.7 (5.5) 6.5 (6.2) 8.2 (8.2) 6.6 (6.3) 1.9 (2.2) 4.7 (4.8) 9.9 (9.9) 3.1 (3.9) 4.9 (4.9) 2.5 (2.3) 5.5 (5.9) 8.0 (8.7)
10.8 (11.1) 5.4 (5.0) 6.3 (5.6) 13.3 (12.5) 7.2 (7.2) 7.8 (7.4) 7.9 (8.4) 7.4 (6.7) 1.6 (1.9) 3.5 (3.2) 9.6 (9.8) 2.4 (3. I) 4.0 (4.4) 3.2 (3.2) 3.2 (2.7) 6.5 (7.9)
u Proteins were hydrolyzed at I 10°C with 5.7 N HCI in the Pica Tag work station and then subjected to amino acid analyses on the Beckman I l9CL ammo acid analyzer. Cysteine and tryptophan were not determined. ’ The proteins (5- I5 pg) were applied to IO% disulfide gels and were recovered by dissolving the gel bands followed by TCA precipitation as described under Materials and Methods. Numbers in parentheses indicate values obtained from the purified proteins not subjected to the gel purification procedure. The results are those obtained from a single analysis of each sample. In samples where multiple analyses were made the results of the additional analyses were not significantly different from one another.
DISULFIDE
GEL
PROTEIN
captoethanol. Thus, it is recommended that solubilizing the gel with dithiothreitol be used as the standard procedure. The ability to obtain peptide maps of unlabeled proteins is greatly enhanced by the use of disulfide crosslinked gels. The reason for this is that several protein bands may be applied in soluble, homogeneous form to a second gel. This eliminates streaking due to slow diffusion of the protein from the gel. Detection of bands on the second gel is facilitated by adequate loads of protein. The application of the disulfide gel purification method to amino acid analysis is facilitated primarily by the fact that the protein is concentrated to a very small volume by the TCA precipitation. This allows the protein to be washed free of contaminating amino acids using minimal amounts of washing solvents (TCA, ethanol, and ether). The contaminating amino acids (predominantly glycine and serine) were increased considerably when the polypeptides were precipitated in the presence of Polybrene. Thus, the most accurate analyses will be obtained when carrier is not included. The removal of all polyacrylamide fragments also prevents accumulation of ammonia, which is a product of acrylamide hydrolysis and which interferes with ninhydrin-based amino acid analyses. The protein may then be hydrolyzed and analyzed by conventional methods to obtain reliable amino acid compositions. The above factors also make feasible the sequencing of proteins isolated by the disulfide gel technique. The TCA precipitation and washing appear to remove contaminants which may interfere with the Edman degradation. The precipitation of the protein also allows one to dissolve it in the small volume (30 ~1) required for application to the gas phase sequencer. In the samples tested thus far the initial and repetitive yields were as high as those obtained from samples purified by conventional methods. Although the limits of sensitivity have not been determined, the method seems workable for sam-
359
ISOLATION
ples in the range 100 to 400 pmol. With the addition of Polybrene as carrier during the precipitation stage it has been possible to obtain sequence information at levels below 100 pmol. Thus, the technique may not be sufficiently sensitive to replace the electroblotting methods of Aebersold et al. (4), Vandekerckhove et al. (3) and Matsudaira ( 16), but should provide a simpler alternative to them when sufficient amounts of sample can be obtained. ACKNOWLEDGMENTS We thank Romie Brown for typing the manuscript and Drs. J. D. Dignam and S. T. Case for helpful suggestions.
REFERENCES
4.
5.
10. I I. 12. 13. 14. 15. 16.
Anderson, P. J. (1985) Anal. Biochetn. 148, 105-I IO. Parekh, B. S., Mehta, H. B., West. M. D., and Montelaro. R. C. (1985) Anul. Biochetn. 148, 87-92. Vandekerckhove, J., Bauw, G., Puype, M., Van Damme. J., and Van Montagu. M. (1985) Ertr. J. Biochem. 152, 9- 19. Aebersold. R. H., Teplow, D. B.. Hood, L. E.. and Kent, S. B. H. (1986) J. Biol. Chem. 261, 4229-4238. Hunkapiller, M. W., and Lujan, E. (1986) in Methods of Protein Microcharacterization (Shively, J. E., Ed.), pp. 89-10 1, Humana Press, Clifton, NJ. Anker. H. S. (1970) FEBS Lets. 7, 293. O’Connell, P. B. H., and Brady, C. J. (1977) Anal. Biochetn. 16, 63-73. Hansen, J. N. (I 976) Anal. Biochetn. 76, 37-44. McConahey. P. J., and Dixon, F. J. (1966) Int. Arch. ANergy 29, 185-189. Rao. S. V. V.. Mamrack, M. D., and Olson, M. 0. J. ( 1982) J. Biol. Chem. 257, 15035-l 504 1. Flyer, D. C., and Tevethia. S. S. (1982) Virology 117.267-270. Laemmli. U. K. (1970) Nature ~London) 227, 680-685. Valenzuela, P., Weinberg, F., Bell, G., and Rutter. N. J. (1986) J. Biol. Chetn. 251, 1464-1470. Lischwe, M. A.. and Ochs, D. (1982) Anal. Biothem. 127,453-457. Lam, K. S.. and Kasper, C. B. ( 1980) Anul. Biothem. 108,220-226. Matsudaira, P. (1987) J. Biol. Chem. 262, 1003510038.
BIOCHEMISTRY
171,352-359
(1988)
Isolation of Proteins for Peptide Mapping, Amino Acid Analyses, and Sequencing Using Disulfide Crosslinked Polyacrylamide Gels’ SEYED H. GHAFFARI,
TAMBA S. DUMBAR, MICHAEL AND MARK 0. J. OLSON~
0. WALLACE,
Department of Biochemistry, The University OfMississippi Medical Center, 2500 North State Street, Jackson, Mississippi 392164505 Received August 28, 1987 Conditions for recovery of small amounts of proteins (l-50 pg) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilixation and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and &galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments. Q 1988 AC&I& yes, IOC. KEY WORDS: sodium dodecyl sulfate-polyacryamide gel electrophoresis; protein precipitation; amino acid analysis; protein sequencing.
Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDSPAGE)3 is a powerful tool in protein biochemistry because of its ability to resolve complex mixtures of polypeptides present at very low concentrations. Recently, methods have been developed for recovery of small quantities of protein from gels for peptide mapping (l), amino acid analyses (2), or protein sequencing (3-5). These techniques involve either electrophoretic elution from gel slices or electrophoretic transfer to nitrocellulose or glass fiber membranes. Although these are generally effective methods for ob’ This work was supported by Grants GM28349, RR 02745, and RR 05386 to M.O.J.O. from the National Institutes of Health. 2 To whom correspondence should be addressed. 3 Abbreviations used: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BAC, N,N’bisacrylylcystamine; Bis, N,N’-methylenebisacrylamide; NCS, N-chlorosuccinimide; PTH, phenylthiohydantoin; TCA, trichloroacetic acid; UHA, urea:water:acetic acid, TEMED, N,N,N,N’-tetramethylethylenediamine. 0003-2697188 $3.00 CopMgbt Q 1988 by Academic Pms, Inc. AI1 rights of reproduction in any form -cd.
taming small quantities of certain proteins, there are a number of difficulties associated with their use: (i) they are cumbersome, time consuming, and require multiple sample manipulations; (ii) they expose the sample to large volumes of solvent, often resulting in contamination; (iii) they require special apparatus; (iv) they often result in low yields of recovery; and (v) they require special pretreatment of the glass fibers onto which the polypeptides are blotted for sequence analyses. Because of these difficulties we have explored an alternative method for isolation of microgram quantities of proteins. A variety of reversible crosslinkers for polyacrylamide gels have been available since the mid- 1970s. These include N,N’-diallyltartardiimide (6), N,N’-dihydroxyethylenebisacrylamide (7), and N,N’-bisacrylylcystamine (8). We have focused on the latter reagent because it is possible to dissolve the gel using relatively mild, neutral, and nonoxidizing conditions. Despite these favorable properties only a few 352
DISULFIDE
GEL
PROTEIN
reports have utilized the disulfide gels for protein isolation. The purpose of this study was to develop methods utilizing the disulfide gel system for isolating microgram or nanogram quantities of protein suitable for use in peptide mapping, amino acid analyses, or protein sequencing. In this paper we describe methods which may be used for all three purposes. MATERIALS
AN5
METHODS
Chemicals and proteins. Most developmental experiments utilized the high-molecular-weight standard mixture from the Sigma Chemical Co. which contained the following proteins (and respective molecular weights): bovine carbonic anhydrase (29,000), egg albumin (45,000), bovine albumin (66,000), rabbit phosphorylase b (97,400), Escherichia coli P-galactosidase (116,000), and rabbit myosin (205,000). In some experiments the molecular weight standard mixture was labeled with ‘25I using the chloramine-T procedure (9). Sperm whale myoglobin (sequencer grade) used in the sequencing studies was obtained from Beckman Instruments. Polyacrylamide, N,N’-methylenebisacrylamide (Bis), N,N’-bisacrylylcystamine (BAC), and N, N, N,N’-tetramethylethylenediamine (TEMED) were purchased from Bio-Rad. Polybrene was purchased from Applied Biosystems, Inc. Partially purified nucleolar protein fractions were obtained from Novikoff hepatoma nucleoli as previously described ( 10). Electrophoresis procedures. Proteins were run on a gel system essentially as described by Flyer and Tevethia (11) using the reversible crosslinker BAC. A 20% stock solution was made with a ratio of acrylamide to BAC of 19.22:0.78 (w/w). The separating gels containing either 7.5 or 10% acrylamide/BAC were made in TBES buffer (to final concentrations of 89 mM Tris-HCl, 89 mM boric acid, 2.8 mM EDTA, 0.1% SDS, pH 8.3) and were thoroughly degassed before polymerization (see Table 1 for composition of gels).
ISOLATION
353
Most gels were cast as 140 X 160 X l-mm slabs in the Bio-Rad Protean I apparatus, although some runs were made in the Mini Protean II system (60 X 80 X 0.75 mm). After pouring, the gels were overlayered with isobutanol. The separating gels were preelectrophoresed in TBES buffer for 3-4 h at 100 V to remove excess reagents and unreacted polymerization products and to equilibrate gels to the proper pH and ionic strength. Due to the presence of mercaptoethanol in sample buffer, stacking gels were of the standard Laemmli type (I 2) containing 5% acrylamide and 0.13% Bis. Samples were applied to the gels with Laemmli-type sample buffer. For evaluation of purity of isolated proteins and peptide mapping, standard Laemmlitype gels were used. After electrophoresis the gels were stained for 1-2 h in 0.1% Coomassie blue in 50% methanol containing 10% acetic acid and destained in a solution containing 10% isopropanol and 10% acetic acid. Isolation of proteins ,from gels. Stained bands were excised, washed for 1 h in water with four changes, cut into small pieces, and transferred to microcentrifuge tubes. Two different procedures for dissolving the gel were used. In the first, l-2 vol of 2-mercaptoethanol plus 1 vol of water (to lower the viscosity) were added. In most cases the gels dissolved within a few minutes. In some instances where the gels did not readily dissolve, the tubes were heated at 80-90°C for 5 min and gently shaken until the gel was liquefied (usually 5-30 min). In the second method, 2 vol of 1 M dithiothreitol in 0.05 M sodium phosphate buffer (pH 7.0) was added to the gel slices, which were then incubated at 70°C for 15 min. Concentrated trichloroacetic acid (TCA) (40%) was added to a final concentration of 15%. In some peptide mapping experiments, polyadenylic acid (poly(A)) carrier was added at a concentration of 40 pg/ml. Also, for some samples subjected to amino acid analyses or sequencing, Polybrene (40 pg/ml) was added prior to the addition of TCA. The tubes were allowed to
354
GHAFFARI
stand at 0-4°C overnight and then centrifuged at 10,OOOg for 30 min in a microcentrifuge at 4°C. The supernatant was decanted and the pellet was washed once as above with 15% TCA. To remove the TCA the pellet was washed twice with ethanol:ether (1: 1, v/v) and once with ether (13). Peptide mapping. In most experiments chemical cleavage was performed before the protein was removed from the gel. Slices containing stained protein bands were washed with 25 ml of water two times for 10 min. For N-chlorosuccinimide (NCS) cleavage the gel slices were subsequently washed two times for 10 min with 10 ml of urea:water:acetic acid ( 1 g: 1 ml: 1 ml; UHA). Then 5 ml of 0.015 M NCS in UHA was added and the cleavage reaction (14) was continued for 1 h at room temperature. For CNBr cleavage the gel slices were washed with water and then with 4 ml of 70% formic acid for 30 min at 25°C. The slices were then incubated with 2 ml of 1% CNBr in 70% formic acid for 1 h at room temperature in the dark. Immediately after the cleavage reactions the slices were washed with water as above, followed by a 20-min wash in running buffer (25 ml) and a 20-min wash in 4 ml of sample buffer which did not contain mercaptoethanol. The slices were then dissolved by adding 20 ~1 (per slice) of sample buffer containing l-5 ~1 of 2-mercaptoethanol. The dissolved slices were heated at 100°C for 3 min and then applied directly to Laemmlitype gels. Other peptide mapping experiments used proteins which had been removed from the gels and precipitated. In these, the cleavage reactions were carried out essentially as above except that reaction volumes were reduced to 50 ~1. After cleavage the samples were diluted with water ( 10 vol) and freezedried before application to the gels. Amino acid analyses and sequencing. The preferred procedure for sample handling prior to hydrolysis was to perform all manipulations in the hydrolysis tubes. With samples for which this could not be done, precip-
ET
AL.
itated proteins were first dissolved in 50 ~1 of 5.7 N HCl (Pierce Sequenal grade) by heating for 5 min at 100°C and transferred to 6 X 50-mm tubes. The samples were dried in a Savant Speed-Vat and subjected to hydrolysis for 22 h at 110°C in a Waters Picotag work station. Amino acid analyses were performed on a Beckman 119CL amino acid analyzer. Protein sequencing was done on an Applied Biosystems 470A sequencer using the 03RPTH program. The precipitated protein samples were dissolved in 30-60 ~1 of l-4% SDS or 70% formic acid and applied to the Polybrene-treated glass fiber filter. PTH amino acids were identified on a Waters HPLC system which was directly interfaced with the sequencer via a Rheodyne 7126 injection valve. In this system approximately 40% of each residue was applied to an Applied Biosystems dedicated PTH amino acid column (2.1 mm) using the solvent system specified by the vendor. RESULTS
Choice of gel electrophoresis conditions, Because of the high resolving power of Laemmli-type gels we attempted a simple substitution of the BAC crosslinker for Bis in initial experiments. This was generally unsuccessful because of difficulties in obtaining the correct level of crosslinking and the inability of certain proteins to enter the gel. Therefore, we adopted the system used by Flyer and Tevethia (1 l), using gels containing either 7.5 or 10% polyacrylamide (Table I). Resolution on this system was adequate for either standard protein mixtures (Fig. 1) or samples of partially purified cell fractions. Furthermore, the use of the Tris-borate buffer system rather than Tris-glycine helped to reduce glycine contamination for amino acid analyses and sequencing (see below). Recovery of proteins from gels. In TCA precipitation of proteins it is a common practice to add a carrier to improve recovery
DISULFIDE TABLE
GEL PROTEIN
1
COMPOSITIONOFPOLYACRYLAMIDESEPARATINGGEL SOLUTIONS WITH BACCROSSLINKER
% Acrylamide 7.5
10
ml of component per 40 ml gel 20% acrylamide/BAC stock” 10x TBES* Hz0 TEMED 10% (NH.,)&Os
15 4 20 1
20 4 14.8 1.2 0.06
0.06
’ Made with a ratio of acrylamide:BAC of 19.22:0.78 (w/w). * 1X TBES contains 89 mM Tris-HCI, 89 mM boric acid, 2.8 mM EDTA, and 0.1% SDS, pH 8.3.
of small amounts of protein. For this purpose we have routinely added poly(A) to the dissolved gel slices prior to TCA precipitation. This has been satisfactory for mapping stud-
ies, but the poly(A) was a source of contamination for amino acid analyses and protein sequencing. It was discovered that reasonable recoveries were possible in the absence of poly(A) or other carriers. The recovered proteins could then be resolubilized in l-4% SDS for further analyses. By gel electrophoresis the recovered proteins had migration characteristics essentially identical to those of the original material and were of high purity (Fig. l), thereby providing the initial evidence for the usefulness of the method. To determine the efficiency of recovery of protein by the TCA precipitation procedure, radioiodinated protein samples were run at various concentrations on the gel system. The stained bands were excised. counted in a gamma counter, dissolved, and precipitated, and the precipitates were counted again. Table 2 indicates that the recovery varied from about 30 to nearly 90%, although most samples were recovered in the range 60-70%. The recovery was dependent on the protein as well as on the concentration of the protein
II EA
BA
1301, C23
Ill
I
B
*
355
ISOLATION
4160K 4C23
,~%* 1 4 160 K 4 130 K 4 C23
FIG. I. Isolation of proteins from disulfide gels. (A) The commercially available high-molecular-weight mixture of proteins (Sigma) was applied to a 10% polyacrylamide gel using N,N’-bisacrylylcystamine (lane I). After electrophoresis the stained bands of bovine serum albumin (BA) and ovalbumin (EA) were cut out, dissolved with mercaptoethanol, precipitated with 10% TCA, and washed with ethanol/ether. The lanes in II show the reelectrophoresis of the precipitated proteins using 10% Laemmli-type gels. (B) Partially purified nucleolar extracts (I and II) were applied to 10% disulfide gels as in A. After electrophoresis the stained bands as indicated were applied to the second gel as in A.
356
GHAFFARI
ET AL.
TABLE 2 RECOVERIESOFPROTEINSFROMSTAINEDBANDS Percentage recovered Micrograms loaded” 50
20 10 5 1
Ovalbumin
Phosphorylase b
/3-Galactosidase
73.2 76.2 61.4 32.5 23.4
62.7 65.7 61.7 68.6 86. I
58.8 75.1 40.2 65.6 56.5
” The radioiodinated proteins were applied to 10% disulfide gels and isolated by TCA precipitation without carrier. The recoveries were calculated from the cpm in each band before the gel was dissolved and the cpm recovered in the pellets after precipitation. Each value is based on duplicate determinations. The range for the duplicates was as much as 10%.
precipitated. For example, the percentage recovery of albumin was considerably lower at low concentrations than at high concentrations. On the other hand, the recovery of phosphorylase b was generally high (about 65%) and less dependent on the concentration of protein in the gel band. It was also found that reasonable recoveries could be expected down to 1 pg of protein. However, recovery of low-molecular-weight proteins was very low; e.g., insulin A or B chains (M, 2300-3400) were recovered in yields of less than 10% (data not shown). On the other hand, apo-myoglobin (Mr 18,360) was recovered in sufficient quantities for sequencing (see below). Thus, the lower limit for effective precipitation would appear to be somewhere between A4,3400 and M, 18,000. Peptide mapping of recovered proteins. Peptide mapping was done with CNBr and NCS on test proteins purified on the BAC gel system. The most convenient procedure was to perform the cleavage reaction in the gel slice before the gel was dissolved. The dissolved slices could then be applied directly to the second gel to yield clearly defined banding patterns (Fig. 2). The advantage of this procedure over mapping methods (14) which employ conventional Bis crosslinked gels is that multiple bands of purified protein can be applied to the same lane of a gel. In ex-
periments not shown when more than two tional gels were applied gel, smearing resulted the cleavage products
CNBr EA BA
it was observed that bands from convento a lane of a second presumably because were removed from
WCS El
BA
FIG. 2. Peptide mapping of proteins isolated from disulfide gels. The commercially available high-molecular-weight mixture of proteins (Sigma) was applied to a 10% polyacrylamide gel using N,N’-bisacrylylcystamine as in Fig. IA. After electrophoresis the stained bands (designated by small letters) were excised, subjected to cleavage with either cyanogen bromide (CNBr) or Nchlorosuccinimdie (NCS), and solubilized as described under Materials and Methods. The cleaved proteins were reelectrophoresed on 12% Laemmh-type gels.
DISULFIDE
GEL
PROTEIN
the first gel slices at variable rates. Dissolving the gel allowed the fragments to form a homogeneous band at the top of the gel prior to separation. In some experiments in which the protein in the stained bands was very dilute and the volume which could be accommodated by the sample slots was limiting, cleavage was done after precipitation. In these (not shown) the cleavage pattern was essentially indistinguishable from the results shown in Fig. 2, although this method was somewhat less convenient. Amino acid analyses of recovered proteins. Several standard proteins were isolated from gel bands by reversing the crosslinking and TCA precipitation. These were subjected to hydrolyses and conventional amino acid analyses. For comparison, samples of the purified proteins obtained commercially were subjected to hydrolyses and amino acid analyses under identical conditions but without further purification. Table 3 indicates that for all proteins analyzed the compositions of the gel-purified proteins were remarkably similar to those of the commercially available preparations. It is not possible to determine whether the minor differences are due to a higher purity of the gel-purified proteins or to minor contaminants introduced by the method. In any event, these results indicated that reliable amino acid compositions could be obtained from gel-purified proteins. Protein sequencing of disuljide gel-purified protein. The final test of the disulfide gel purification method was to perform protein sequencing on a standard protein. For this purpose, whale myoglobin (approximately 20 pg) was applied to the gel system, purified, and precipitated without carrier. Heating the precipitated sample at 100°C for 3 min in 1% SDS solubilized about 70% of the protein as measured by amino acid analysis. We also found 70% formic acid to be an effective solvent, giving nearly complete recovery without heating the sample. An aliquot of the solubilized protein was applied to a precycled glass fiber filter of the gas phase sequencer. The results of 12 cycles of Edman
357
ISOLATION
degradation were comparable to results obtained from commercially purified standards; the initial yield was 9 1% and the repetitive yield was 93.5%. This is well within the range specified by the manufacturer for the test protein; the repetitive yield should be at least 92% and 94% for 100 pmol and 1 nmol of myoglobin, respectively. Although the runs were not carried beyond cycle 13, the repetitive yield suggests that extensive Edman degradations could be carried out on this material. Furthermore, the level of contaminants was no higher than that in runs made with conventionally purified proteins, although the glycine and proline background was 2-4 pmol higher than normal. Thus, the method appears to be useful for purification of small amounts of proteins or peptide for amino-terminal sequence analyses. We have successfully applied this technique to several polypeptides of known and unknown sequence, e.g., P-galactosidase and CNBr fragments from nucleolar protein C23. For sequencing we have found that addition of Polybrene (1) as a carrier aids in recovery and transfer of the protein to the filter disk of the sequencer. DISCUSSION
The data presented in this paper indicate that by altering the crosslinking agent a polyacrylamide gel electrophoresis system can be used to purify proteins for peptide mapping, amino acid analyses, and sequencing. This method, which provides an alternative to electrotransfer and electroelution methods, utilizes ordinary electrophoretic and laboratory equipment. A possible disadvantage is that the resolving power of the gel system used in these experiments is slightly less than that of Laemmli-type systems. Using the 7.5 and 10% acrylamide systems discussed in this paper, resolution was adequate for polypeptides between M, 40,000 and A4,200,000; below M, 40,000 the band spreading would require a prefractionation step to make the starting material less complicated. The reso-
358
GHAFFARI
lution may also be decreased by interaction of sulthydryl groups in the protein with the gel matrix, although we have not systematically examined this. Thus, for highly complex protein mixtures, reduction and carboxymethylation may be required. In the attempts to adapt the disulfide gel system to the three techniques discussed, some difficulties were encountered. First, it was necessary to adjust the concentrations of ammonium persulfate and TEMED to control the rate of polymerization. The amounts indicated in Table 1 were determined using the guidelines outlined in Bio-Rad Technical Bulletin 2045. These concentrations have been satisfactory for several batches of BioRad chemicals, but adjustments may be necessary for other batches or for chemicals from other vendors.
ET AL.
The second problem was the tendency of protein to stick at the interface between the stacking and the separating gel. This was solved by preelectrophoresis of the gel immediately after polymerization to remove the reactants and charged reaction products. The final problem was that of solubilizing the gel containing the stained protein band. Solving the first two problems largely solved this problem by preventing formation of covalent crosslinks. However, it was also necessary to maintain an adequate ratio of the volume of mercaptoethanol to the volume of the gel slice. At least a 2: 1 ratio was required for the gel slices to dissolve and for the viscosity of the solution to be low enough for TCA precipitation to take place. We also found that 1 M dithiothreitol was a much more effective solubilizing agent than mer-
TABLE 3 AMINO
ACID
COMPOSITIONS
OF GEL-PURIFIED
AND
CONTROL
PROTEINS
mol % Amino acid“ Asx Thr Ser GIX
Pro GlY Ala Val Met Be Leu TY~ Phe His LYS A%
Bovine albuminh 9.0 (9.5) 5.1 (5.4) 4.6 (5.7) 16.8 (14.1) 5.8 (7.1) 4.1 (2.8) 7.7 (7.9) 5.4 (7.2) 0.9 (0.6) 2.8 (0.6) 17.2 (15.7) 2.0 (2.8) 4.3 (4.9) 2.3 (2.3) 7.9 (10.2) 4.2 (4.4)
Ovalbumin 9.7 (10.1) 3.7 (4.2) 9.0 (9.3) ll.9(12.1) 5.7 (4.3) 7.7 (6.2) 8.6 (9.4) 8.1 (8.9) 2.3 (3.2) 4.7 (4.8) 9.1 (8.6) 1.9 (1.9) 5.2 (5.0) 2.1 (1.6) 5.3 (5.8) 4.9 (4.8)
Phosphorylase b
P-Galactosidase
11.3 (11.7) 4.5 (4.2) 4.3 (2.9) 12.5 (12.5) 5.7 (5.5) 6.5 (6.2) 8.2 (8.2) 6.6 (6.3) 1.9 (2.2) 4.7 (4.8) 9.9 (9.9) 3.1 (3.9) 4.9 (4.9) 2.5 (2.3) 5.5 (5.9) 8.0 (8.7)
10.8 (11.1) 5.4 (5.0) 6.3 (5.6) 13.3 (12.5) 7.2 (7.2) 7.8 (7.4) 7.9 (8.4) 7.4 (6.7) 1.6 (1.9) 3.5 (3.2) 9.6 (9.8) 2.4 (3. I) 4.0 (4.4) 3.2 (3.2) 3.2 (2.7) 6.5 (7.9)
u Proteins were hydrolyzed at I 10°C with 5.7 N HCI in the Pica Tag work station and then subjected to amino acid analyses on the Beckman I l9CL ammo acid analyzer. Cysteine and tryptophan were not determined. ’ The proteins (5- I5 pg) were applied to IO% disulfide gels and were recovered by dissolving the gel bands followed by TCA precipitation as described under Materials and Methods. Numbers in parentheses indicate values obtained from the purified proteins not subjected to the gel purification procedure. The results are those obtained from a single analysis of each sample. In samples where multiple analyses were made the results of the additional analyses were not significantly different from one another.
DISULFIDE
GEL
PROTEIN
captoethanol. Thus, it is recommended that solubilizing the gel with dithiothreitol be used as the standard procedure. The ability to obtain peptide maps of unlabeled proteins is greatly enhanced by the use of disulfide crosslinked gels. The reason for this is that several protein bands may be applied in soluble, homogeneous form to a second gel. This eliminates streaking due to slow diffusion of the protein from the gel. Detection of bands on the second gel is facilitated by adequate loads of protein. The application of the disulfide gel purification method to amino acid analysis is facilitated primarily by the fact that the protein is concentrated to a very small volume by the TCA precipitation. This allows the protein to be washed free of contaminating amino acids using minimal amounts of washing solvents (TCA, ethanol, and ether). The contaminating amino acids (predominantly glycine and serine) were increased considerably when the polypeptides were precipitated in the presence of Polybrene. Thus, the most accurate analyses will be obtained when carrier is not included. The removal of all polyacrylamide fragments also prevents accumulation of ammonia, which is a product of acrylamide hydrolysis and which interferes with ninhydrin-based amino acid analyses. The protein may then be hydrolyzed and analyzed by conventional methods to obtain reliable amino acid compositions. The above factors also make feasible the sequencing of proteins isolated by the disulfide gel technique. The TCA precipitation and washing appear to remove contaminants which may interfere with the Edman degradation. The precipitation of the protein also allows one to dissolve it in the small volume (30 ~1) required for application to the gas phase sequencer. In the samples tested thus far the initial and repetitive yields were as high as those obtained from samples purified by conventional methods. Although the limits of sensitivity have not been determined, the method seems workable for sam-
359
ISOLATION
ples in the range 100 to 400 pmol. With the addition of Polybrene as carrier during the precipitation stage it has been possible to obtain sequence information at levels below 100 pmol. Thus, the technique may not be sufficiently sensitive to replace the electroblotting methods of Aebersold et al. (4), Vandekerckhove et al. (3) and Matsudaira ( 16), but should provide a simpler alternative to them when sufficient amounts of sample can be obtained. ACKNOWLEDGMENTS We thank Romie Brown for typing the manuscript and Drs. J. D. Dignam and S. T. Case for helpful suggestions.
REFERENCES
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10. I I. 12. 13. 14. 15. 16.
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