The recovery of nitrocellulose-bound protein

ANALYTICAL

BIOCHEMISTRY

148, 105-110 (1985)

The Recovery

of Nitrocellulose-Bound

Protein

P.J. ANDERSON Department of Biochemistry, University of Ottawa, Ottawa, Ontario KIH 8M5. Canada Received March 1, 1985 Conditions for the electroelution of protein from polyacrylamide gels to nitrocellulose and its subsequent recovery have been examined. A procedure is described whereby soluble material suitable for further analysis can be obtained. Nitrocellulose-bound protein is dissolved in acetone. It can be precipitated from solution in the presence of a carrier such as Polybrene in a form that can subsequently be solubilized in good yield. 8 1985 Academic PIW., 1~. KEY WORDS: nitrocellulose; protein recovery.

Electroelution of proteins from polyacrylamide gels and adsorption on an immobilizing layer such as nitrocellulose is an increasingly popular technique for subsequent analysis of proteins separated by electrophoresis on polyacrylamide gels. The principles of the procedure, known as protein blotting, have recently been reviewed (1). Nitrocellulose membranes are widely used in protein blotting because proteins are absorbed to the membranes under conditions of low ionic strength which facilitates electroelution from gels. The mechanism of interaction between the proteins and the nitrocellulose is uncertain (2) but does not require the formation of covalent bands. Despite this, proteins are firmly bound, which allows incubation in a variety of buffers (1) without removal of protein from the nitrocellulose. This property has proved to be very useful in the application of overlay techniques to protein identification. However, in many cases it would be of value to recover the protein from the nitrocellulose. The present report describes a method for removal of protein from nitrocellulose in a form which can be used for further protein analysis. METHODS

AND

MATERIALS

powders of rabbit back muscle (4). Tubulin was prepared from fresh calf brain (5). Ribonuclease A from bovine pancreas, bovine serum albumin, egg white lysozyme, chymotrypsin, and materials for making polyacrylamide gels were obtained from Sigma Chemical Company. Ethyl acetimidate, guanidine hydrocholoride, and Polybrene were obtained from Pierce Chemical Company. Iodo[2-‘4C]acetic acid, ethyl[ l-‘4C]acetimidate, and [ “C]formaldehyde were obtained from New England Nuclear. Chemical modifications were carried out on proteins denatured in guanidine hydrocholoride using radiolabels diluted with carrier to approximately 5 mCi/mmol. Iodoacetic acid was used to modify sulfhydryl groups (6). Amino groups were modified by amidination (7) or by reductive methylation (8). Electrophoresis was carried out on 1.5mm-thick sodium dodecyl sulfate (SDS)‘containing 7.5% polyacrylamide slab gels (9). Band widths were either 0.5 or 1 cm. Electroelution onto Bio-Rad nitrocellulose was performed in a Bio-Rad Trans-Blot cell in 25 mM Tris, 192 mM glycine, 20% methanol. Protein was located by staining with amido black. Radioactivity was determined by liquid scintillation counting of 50-~1 aliquots.

Myosin was isolated from rabbit back muscle (3). Actin was isolated from acetone

’ Abbreviation used: SDS, sodium dodecyl sulfate. 105

0003-2697/85 $3.00 Copyright 0 1985 by Academic F%s. Inc. All rights of reproduction in any form resewed.

106

P. J. ANDERSON

Peptide maps were prepared on Eastman chromagram cellulose sheets and autoradiography was used to locate radioactivity. Amino acid analysis was carried out on a modified Varian Vista 54 liquid chromatograph (10). An Eppendorf microcentrifuge set for 15,000 rpm was used for centrifugation.

counts applied was soluble after acetone solubilization of nitrocellulose-bound proteins and those counts were not precipitated after centrifugation in an Eppendorf microcentrifuge at 15,000 rpm for 5 min. Formic acid soluble counts varied with the protein used. Table 1 summarizes data for six proteins. The low-molecular-weight proteins ribonuclease and lysozyme were obtained in good yield by extraction with formic acid. Since formic acid is volatile and the nitrocellulose was not dissolved by the formic acid, ribonuclease and lysozyme can be recovered from nitrocellulose in good yield in a form suitable for further analysis by formic acid extraction. The other proteins were recovered in much lower yield in 70% formic acid and still lower yields were obtained when 98% formic acid was used. However, these proteins were soluble in acetone when dissolved bound to nitrocellulose. For subsequent protein analysis such as peptide mapping or amino acid sequencing, it is necessary to recover the proteins from acetone free of nitrocellulose. It was found that the addition of water or a 0.5% solution of ammonium bicarbonate to the acetone-solubilized nitrocellulose-bound proteins could cause the proteins to become insoluble. The amount of aqueous solution

RESULTS

Initial experiments to determine conditions for recovery of protein from nitrocellulose were carried out on proteins chemically modified by reductive methylation with [‘4C]formaldehyde. Proteins were dissolved in 70% formic acid and 2.5~~1 aliquots containing approximately 5000 counts and 5 pg protein were spotted onto nitrocellulose. After air-drying, the nitrocellulose bound protein was stained in amido black and destained in methanol:acetic acid:water (25:7:67). Stained areas of approximately 10 mm* were cut out and mixed with 1 ml of formic acidwater (70:30) or 1 ml of reagent-grade acetone. The nitrocellulose did not dissolve in the formic acid but completely dissolved in acetone. After 30 min, soluble radioactivity was determined by counting aliquots of 50 ~1. For all proteins examined, in excess of 95% of TABLE S~LUBILIZATION

1

ANDRECOVERYOFNITROCELLULOSE-BOUND

PROTEINS

Ratio of counts recovered from acetone to counts recovered by formic acid extraction

Protein

Ratio of formic acid to acetone soluble counts

No carrier

Polybrene

Ribonuclease Lysozyme Albumin Actin Tubulin Myosin

0.90 0.81 0.23 0.09 0.33 0.17

0.46 0.71 0.67 0.79 1.24 1.33

0.12 1.00 2.54 4.52 1.42 1.58

Albumin 0.80 1.01 1.56 4.7 1 1.90 1.52

Note. Proteins were made radioactive by reductive methylation in the presence of [‘4C]formaldehyde. Aliquots of the labeled proteins dissolved in 70% formic acid were spotted in duplicate on nitrocellulose and airdried. Nitrocellulose-bound protein was extracted with 70% formic acid or solubilized in acetone. Protein solubilized in 1 ml acetone was recovered in 70% formic acid after precipitation by the addition of 150 ~1 0.5% ammonium bicarbonate in the absence or presence of the carriers Polybrene (2 mg/ml) or bovine serum albumin (2 mg/ml).

ELECTROPHORETIC

NITROCELLULOSE-BOUND

added was critical. All counts could be removed from solution by 5 min centrifugation in an Eppendorf microcentrifuge after addition of aqueous solution to acetone-solubilized nitrocellulose-bound protein to a final concentration of between 13 and 15% (v/v). Less aqueous solution resulted in incomplete precipitation of counts. More aqueous solution resulted in coprecipitation of large amounts of nitrocellulose. A proportion of the counts precipitated under optimum conditions could be resolubilized in 70% formic acid free of nitrocellulose. Table 1 indicates that for all proteins examined, comparable or much better yields of protein free of nitrocellulose can be obtained by precipitation from acetone and subsequent solubilization in formic acid compared to formic acid extraction. The presence of carriers, either Polybrene or bovine serum albumin, in the aqueous solutions added to acetone significantly improves yields. Under the conditions used they are both comparably effective. Two proteins whose recovery from nitrocellulose was significantly improved by precipitation from acetone were actin and tubulin. The conditions for recovery of these proteins after electrophoresis and nitrocellulose blotting were examined in greater detail.

PROTEIN

RECOVERY

107

pg protein applied to polyacrylamide gel FIG. 1. Recovery of protein on n&cellulose. Amounts of tubulin (0) or actin (0) as indicated were applied to single wells of a polyacrylamide slab gel and, after electrophoresis, transferred to nitrocellulose by electroelution. Protein bands on nitrocellulose were located by staining with amido black and then dissolved in acetone. Counts were determined by scintillation counting of aliquots of acetone solutions. Actin was labeled with iodo[2-14C]acetic acid and tubulin was acetimidated with ethyl1 I-‘4C]acetimidate.

Initial experiments were designed to maximize the yield of nitrocellulose-bound protein. Electroelutions of radioactive proteins separated by SDS-polyacrylamide gel electrophoresis onto nitrocellulose were carried out for various lengths of time. The proteins were located on electroblots by staining with amido black and the protein bands were dissolved TABLE 2 in 1 ml of acetone. Table 2 summarizes recoveries calculated from the amounts of RECOVERYOFCOUNTSONNITROCELLULOSE AS A FUNCTIONOF TIME OFELECTROELUTIONAT~O V radioactivity detected in acetone-solubilized nitrocellulose-bound protein. It is apparent Recovery(%+SD) that protein is rapidy adsorbed to the nitroElectrocellulose under the conditions used. Losses 1 elution (Strip A) (Stti; B) 2 4 appear to be due to protein passing through (W: the nitrocellulose layer as seen from the 58 f 7 28 + 1 50 -c 8 38 f 8 counts adsorbed on a second layer of nitroNote. Actin was labelled with iodo[2-‘4C]acetic acid cellulose. From these experiments a standard and subjected to eletrophoresis in SDS-containing poly- blot of 1 h at 50 V was adopted in which acrylamide gel prior to electroelution onto nitrocellulose. close to 60% of actin or tubulin counts was In the l-h blot two strips of nitrocellulose (A and B) recovered in single-layer nitrocellulose bIots. were used. Strip A was closest to the polyacrylamide gel. Figure 1 indicates that over a wide range Protein was located on nitrocellulose by staining with of amounts of protein applied, the proportion amido black. Protein bands were cut out and dissolved recovered by electroblotting was constant. in acetone and aliquots were counted in Aquasol. Four separate protein bands were analyzed from each blot. The recovery appears to be independent of

108

P. J. ANDERSON

the type of chemical modification and similar for both actin and tubulin. The effect of increasing the amount of protein in polyacrylamide gel separations was to increase the area occupied by the protein band in the electroblot. This effect was examined in more detail by the dilution of aliquots of actin labeled with ethyl[ l-‘4C]acetimidate with increasing amounts of actin labeled with the nonradioactive form of the same reagent. Electroblots of these mixtures after separation by polyacrylamide gel electrophoresis indicated that there was no loss in counts recovered over the range 2.5 to 50 pg protein applied per lane. When 100 I.cg protein was applied, the radioactivity recovered decreased by about 20%. With loads of from 2.5 to 100 pg protein, the area of the stained spot corresponding to actin on the nitrocellulose increased from 0.14 to 0.42 cm’. To examine conditions for protein recovery from nitrocellulose, actin separated by electrophoresis and bound to nitrocellulose by electroblotting was precipitated from acetone solutions. To 1 .O ml acetone-solubilized protein, 0.15 ml 0.5% ammonium bicarbonate containing carrier Polybrene or bovine serum albumin was added and the protein was precipitated by centrifugation in an Eppendorf microcentrifuge for 5 min at 15,000 t-pm. Two methods were used to attempt to redissolve the precipitated protein. In the first of these the precipitated material was extracted with 70% formic acid overnight. In the second procedure the precipitated material was digested with 5 &ml chymotrypsin in 0.5% ammonium bicarbonate at 37°C overnight. After centrifugation the fraction of counts redissolved was determined by scintillation counting of aliquots of the supernatant. Table 3 shows that with either bovine serum albumin (2 mg/ml) or Polybrene (2 mg/ml) present in the 0.5% ammonium bicarbonate solutions used to precipitate the protein, more than 50% of the precipitated counts could be recovered in soluble form. The amount of carrier did not appear to be

TABLE 3 SOLUBILIZATION

OFNITROCELLULOSE-BOUND

PROTEIN

Carrier

Solubilization procedure

solubilized (% + SD)

counts

Bovine serum albumin Bovine serum albumin Polybrene Polybrene

70% formic acid

49 + 4

Chymottyptic digestion 70% formic acid Chymotryptic digestion

56 k 2 58 * 8 53 + 7

Note. Nitrocellulose bands of actin which had been acetimidated with ethyl[ 1-“‘C]acetimidate were precipitated from l-ml acetone solutions by the addition of 0.15 ml of 0.5% ammonium bicarbonate containing either 2 mg/ml bovine serum albumin or 2 mg/ml Polybrene. After centrifugation, the precipitated material was redissolved in 250 pl formic acid or by digestion with 250 ~1 chymotrypsin in ammonium bicarbonate as described in the text. Soluble counts were determined for three protein bands per treatment by liquid scintillation counting.

critical. Similar recoveries were observed with carrier concentrations between 1 and 10 mg/ ml in the 0.5% ammonium bicarbonate solution. Figure 2 shows that further protein analysis, such as peptide mapping, can be carried out on protein solubilized from nitrocellulose. The figure is an autoradiograph of chymotryptic peptides produced from actin carboxymethylated with iodo[2-14C]acetic acid. The peptides were produced from material separated on polyacrylamide gels and recovered from nitrocellulose by precipitation from an acetone solution in the presence of Polybrene and subsequently digested with chymotrypsin. The map was identical to a map generated from the labeled actin prior to electrophoresis and electroblotting. Amino acid compositions of proteins recovered from nitrocellulose blots in the presence of Polybrene by extraction with formic acid were determined after hydrolysis overnight in 6 N HCI. Actin and tubulin compositions were unaltered by the isolation procedures except for an apparent increase

ELECTROPHORETIC

NITROCELLULOSE-BOUND

PROTEIN

RECOVERY

109

FIG. 2. Autoradiograph of chymotryptic peptides of actin carboxymethylated with iodo[2-“‘Clacetic acid and solubilized from nitrocelhtlose. Separation of peptides in the first dimension was by electrophoresis in pH 2.1 buffer (acetic acid:formic acid:water, 8:2:90). Chromatography in the second dimension was in butanokacetic acid:water:pyridine ( I5:3: 12: 10). Origin (0) and mobility (m) of the colored marker, cdinitrophenyl lysine.

in glycine content of blotted proteins. This may be due to residual glycine remaining from the Tris-glycine blotting buffer. DISCUSSION

The two low-molecular-weight proteins examined, ribonuclease and lysozyme, could be obtained in good yield from nitrocellulose by extraction with formic acid. Formic acid is volatile and leaves the nitrocellulose intact so it provides a useful method for recovering proteins. However, four of the proteins used in the present study were not readily dissociated from nitrocellulose by formic acid. An alternate procedure for recovering these proteins in good yield, which is also applicable

to the recovery of ribonuclease and lysozyme and therefore appears to be of general utility, has been developed. All proteins examined were soluble in acetone when dissolved bound to nitrocellulose. Residual nitrocellulose bound to protein after solubilization in acetone may have an emulsifying effect on the proteins. The addition of dilute ammonium bicarbonate in certain portions to the acetonesolubilized proteins renders the proteins insoluble while leaving the bulk of dissolved nitrocellulose in solution. All proteins tested were completely insoluble in a 1:6 mixture of 0.5% ammonium bicarbonate and acetone and could be removed from such mixtures by centrifugation. Since proteins precipitated in this way could only be partially recovered

110

P. J. ANDERSON

in 70% formic acid, it is likely that some nitrocellulose remains associated with the protein throughout solubilization and subsequent precipitation. However, this procedure, particularly in the presence of the carriers Polybrene or bovine serum albumin, results in release of substantially more protein bound to nitrocellulose than formic acid treatment for a variety of tightly bound proteins. Conditions for application of this procedure for two proteins, actin and tubulin, separated by polyacrylamide electrophoresis and electroblotted onto nitrocellulose, have been maximized. Actin, a protein which appears to be particularly strongly associated to nitrocellulose on the basis of recovery in formic acid, can be obtained in good yield after electrophoresis and electroblotting. The protein is apparently unaltered structurally on the basis of amino acid analysis and peptide mapping when precipitated from acetonesolubilized nitrocellulose blots in the presence of Polybrene. The use of this carrier should be particularly beneficial in application of the method to nitrocellulose-bound proteins requiring further structural analysis since Polybrene facilitates automated sequence analysis in both spinning cup (11) and gasliquid-phase ( 12) sequencers.

ACKNOWLEDGMENT This work was supported by the Medical Research Council of Canada. REFERENCES 1. Gershoni, J. M., and Palade, G. E. (1983) Anal. Biochem. 131, l-15. 2. Wallis, C., Melnick, J. L., and Gerba, C. P. (1979) Annu. Rev. Microbial. 33, 413-437. 3. Lowey, S., Slayter, H. S., Weeds, A. G., and Baker, H. (1969) J. Mol. Biol. 42, l-29. 4. Spudich, J. A., and Watt, S. (1971) J. Biol. Chem. 246,4866-487

1.

5. Shelanski, M. C., Gaskin, F., and Cantor, C. (1973) Proc. Natl. Acad. Sci. USA 70, 765-768. 6. Anderson, P. J., and Randall, R. F. (1975) Biochem. J. 145, 575-579. 7. Dubois, G. C., Robinson, E. A., Inman, J. T., Perham, R. N., and Appella, E. (I 98 1) Biochem. J. 189, 335-340. 8. Jentoft, N., and Dearborn, D. G. (1979) J. Biol. Chem. 254,4359-4365. 9. Weber, K., and Osborne, M. (I 969) J. Biol. Chem. 244, 4406-44

12.

IO. Klapper, G. (I 982) in Methods in Protein Sequence Analysis (Elzinga, M., ed.), pp. 509-5 15, Humana Press, Clifton, N. J. 11. Hunkapiller, M. W., and Hood, L. E. (1978) Biochemistry 17, 2124-2133. 12. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dreyer, W. J. (198 1) J. Biol. Chem. 256, 7990-7997.