A general immunochemical method for detecting proteins on blots

ANALYTICAL

BIOCHEMISTRY

137,

2 1o-2 16 ( 1984)

A General lmmunochemical

Method for Detecting

Proteins on Blots

JASON M. KITTLER, NATALIE T. MEISLER, DACE VICEPS-MADORE. JOHN A. CIDLOWSKI,’

AND JOHN W. THANASSI~

Depurtment 01‘Biochemistry, University qf Vermont College of Medicine. Burlington, Vermont 05405 Received

August

18, 1983

Following horizontal electroelution, or blotting, of proteins from polyacrylamide gels to immobilizing matrices, such as nitrocellulose or Zeta-bind paper, the transferred proteins can be derivatized in .situ with pyridoxal 5’-phosphate and sodium borohydride. After a quenching step to eliminate nonspecific binding of antibody to the protein-binding matrix, the blot is incubated with a solution containing a mouse monoclonal antibody specific for the 5’-phosphopyridoxyl group. The transferred proteins can then be located on the blot with second antibody staining procedures employing either a peroxidase-linked goat anti-mouse F(ab’h antibody or a peroxidaselinked avidin/biotin system. The solid-phase enzyme-linked immunosorbent assay method described in this report is a mild, general, and sensitive immunochemical method for the detection of proteins on protein-binding matrices. KEY WORDS: protein blotting; anti-phosphopyridoxyl antibodies; nitrocellulose; electrophoresis: immune assays: monoclonal antibodies.

Blotting is defined as “the processof transferring macromolecules from gels to an immobilizing matrix” ( 1). Such procedures have become valuable and widely used methods in the study of macromolecules and in the analysis of their functions. The initial study involving protein blotting, as opposedto nucleic acid blotting, was published by Towbin ef al. in 1979 (2). Since that time, a large number of studies which employ the experimentally simple technique of protein blotting has been published. A recent review on the principles and applications of protein blotting discusses the advantages as well as the range of applications of these procedures ( I). The most commonly used high-resolution, protein-immobilizing blotting matrix is nitrocellulose (NC)? paper which interacts with ’ Present address: Departments of Physiology. Biochemistry and Nutrition. University of North Carolina. Chapel Hill, N. C. 27514. 2 To whom correspondence should be addressed. 3 Abbreviations used: NC. nitrocellulosc; ZB, Zeta-bind; PLP. pyridoxal 5’-phosphate; PPxy-, 5’-phosphopyridoxyl; SDS. sodium dodecyl sulfate; PBS-X. 10 mM phosphate-

proteins in a compiex fashion (3). Zeta-bind (ZB), a positively charged nylon membrane, has been reported to have certain advantages over NC paper such asgreater protein-binding capacity and mechanical strength (4). A limitation in protein blotting procedures centers around the currently available protein detection methods. For example, amido black is not sufficiently sensitive and silver stain is difficult to use on NC blots. For ZB blots, there is at the present time no satisfactory method for the detection of proteins (4). We have developed a general method for the detection of proteins on blots. It depends on the use of pyridoxal S-phosphate (PLP) and sodium borohydride to derivatize matriximmobilized proteins. The resulting S-phosphopyridoxyl (PPxy) proteins can then be located by solid-phase, double-antibody enzyme-linked immunosorbent methods in which the first antibody is a mouse monoclonal antibody directed against the PPxy ___~__~~~~~ ~~~~~ ~~ buffered saline, pH 8; PBS-7.3. 10 mM phosphate-buffered saline. pH 7.3; BSA. bovine serum albumin.

IMMUNOCHEMICAL

DETECTION

group (5,6). The method is conceptually similar to one recently reported by Wojtkowiak et al. (7) who derivatized blotted proteins with 2,4-dinitrofluorobenzene and detected the resulting dinitrophenyl-proteins by an immunolocalizaticln method which employs antibodies to the dinitrophenyl group. MATERIALS

AND

METHODS

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of liver extracts and transfer to NC paper (pore size. 0.15 pm; Schleicher and Schuell) were performed as described previously (5,6). Resolved proteins on gels were detected by Coomassie blue stain or silver stain (8). Proteins on NC blots were visualized by staining with amido black or by either of the immunochemical procedures described below. Rat liver nuclei were prepared by the method of Chiu et ~1. (9) with minor modifications. Chromatin was isolated as described by Pumo et ~11.(10) and prepared for electrophoresis according to the procedure of Glass et al. (1 l), except that the chromatin samples were applied to the gel in equal volumes of a solution containing 0. I% SDS, 0.1 %I 2-mercaptoethanol, and 8 M urea in 0.1 M sodium phosphate buffer (pH 7.0) with bromphenol blue as the tracking dye. Gels were run for approximately I9 h at 70 mA at ambient temperatures according to the procedure of Prince and Campbell ( 12), which was adapted from a procedure described by Wilhelm cl uI. ( 13). Chromatin was fractionated by the method of Chiu et ~1. (9) as modified by Prince and Campbell ( 12) into an H 1-containing fraction and a fraction containing histones other than H I. Histone:< were prepared for electrophoresis as described above for chromatin. SDS-gel electrophoresis-resolved chromatin and histone samples were horizontally electroeluted to either NC or ZB (Bio-Rad ZetaProbe) with modifications of methods previously used in our laboratory (5.6). Specifically, the modifications included three 20-min soakings of the polyacrylamide gels prior to their transfer to the immobilizing matrices.

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The soaking medium was the electroelution buffer of Towbin et al. (2) with the pH adjusted to 8.8 with sodium hydroxide. Electroelutions were performed at 400 mA (8 h, 4°C) using a Bio-Rad Trans-Blot cell. Derivatization of matrix-immobilized pmteins to PPx)‘-proteins. After electroelution of proteins from gels to NC or ZB papers, the blots were washed in 10 mM phosphate-buffered saline, pH 8.0 (PBS-S), to remove electroelution buffer. The blots were then incubated with shaking at room temperature for 20 min in a freshly prepared. nitrogen-gassed solution of 0.3 mM PLP (Sigma) in PBS-8 (pH readjusted to 8.0 with sodium hydroxide). After being washed in PBS-S, the blots were incubated with shaking (30 min. room temperature) in a freshly prepared solution of sodium borohydride in PBS-8 (300 mg/125 ml). The blots now contained PPxy-proteins. Excess borohydride was removed by five washes with PBS-7.3 (5 min per wash, room temperature). Quenching procedurr~s. It is necessary to block or quench the immobilizing matrices in order to eliminate nonspecific binding of specific antibodies. The qtienching procedure for nitrocellulose blots has been described elsewhere (5,6). The quenching procedure for ZB blots requires longer incubation times and more concentrated solutions of blocking proteins (1,4). Accordingly. the blocking solution for ZB blots contained 10% bovine serum albumin (BSA, fraction V, Sigma’) and 2.5% human plasma in PBS-7.3. The human plasma had been previously dialyzed against 5 rnM hydroxylamine in PBS-7.3 to remove endogenuus PLP (5). ZB blots were quenched for 12 h at 45°C. Quenched blots were washed three times in PBS-7.3 (5 min per wash). Imnl~mok#~u(i~ati~)n procedwc~.~. Two general methods were employed, each using as first antibody a mouse monoclonal anti-PPxy antibody (5). One method has been described previously (5.6) and uses a commercially available second antibody. horseradish peroxidasc-conjugated goat anti-mouse F(ab’), (Cappel). The second method uses as second antibody a I: IO.,000 dilution (in 1% BSA in

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PBS-7.3) of a biotin-conjugated F(ab’), fragment of goat anti-mouse IgG (H and L chains) (Cappel). Blots are exposed to this solution for 1 h at 37°C followed by several hours at 4°C. After washing (three times, 10 min per wash) in 0.05% Tween 20 in PBS-7.3, horseradish peroxidase-conjugated avidin (Cappel), diluted 1: 10,000 in 1% BSA in PBS-7.3, is added. The blot is incubated in this solution for 90 min at 37°C then overnight at 4°C. followed by washing with 0.05% Tween 20 in PBS-7.3. Blots are visualized using a diaminobenzidine-hydrogen peroxide staining solution (5,6). RESULTS

Liver cytosolicproteins. Proteins in liver cytosolic extracts were separated electrophoretically on 7.5/0.3% acrylamidejbisacrylamide running gels with a 5/0.2% stacking gel. The resolved proteins were detected on gelsby silver and Coomassieblue stains. NC blots were visualized by amido black. PPxy-derivatized proteins were detected on NC blots by the avidin/biotin-dependent immunochemical

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staining procedure described under Materials and Methods. ZB blots were visualized by the alternate immunochemical procedure which uses a horseradish peroxidase-linked second antibody to the first antibody, a mouse monoclonal antibody to the PPxy group (see Materials and Methods and Refs. (5) and (6). Representative results are provided in Fig. 1. It is apparent from Fig. 1 that the immunoblot procedure that we have developed for detecting PPxy-proteins on NC blots is at least as sensitive as the corresponding silver stain on gels and considerably more sensitive than amido black staining of NC blots. Although the sensitivity of the anti-PPxy immunochemical detection method on the ZB blot shown in Fig. 1 is not as great as on the NC blots, it represents,to our knowledge, the only general method for detecting proteins on ZB blots. Chronzatin and histones. Rat liver chromatin and histones were separated electrophoretically on 10/0.3% acrylamide/bisacrylamide running gelswith a 610.2% stacking gel. These concentrations were chosen to determine the capabilities of the immunochemical

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FIG. I. SDS-gel electrophoresis of liver cytosolic extracts. Samples containing the indicated microgram amounts of protein, as determined by the Lowry method (35). were electrophoresed and stained directly with silver stain or Coomassie stain as indicated. NC blots were stained directly with amido black as shown. or after derivatization of matrix-bound proteins with PLP and sodium borohydride followed by the immunoblot procedure using monoclonal anti-PPxy antibodies and the avidin/biotin system described under Materials and Methods. The lane marked ZB used Zeta-hind instead of NC as the immobilizing matnx and was stained after derivatization with PLP and sodium borohydride followed by the immunoblot procedure using monoclonal anti-PPxy antibody and horseradish peroxidase linked-goat anti-mouse F(ab’j2 second antibody (see Materials and Methods). Molecular weights are indicated at the left edge of the figure.

IMMUNOCHEMICAL

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DETECTION

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FIG. 2. SDS-gel electrophoresis of rat liver chromatin. Samples containing the indicated microgram amounts of DNA. as determined by absorbance at 260 nm. were electrophoresed and stained with Coomassie stain. NC blots were stained directly with amido black, or after derivatization of matrix-bound proteins with PLP/sodium borohydride followed by the immunoblot procedure using monoclonal anti-PPxy antibody and horseradish peroxidase-linked goat anti-mouse F(ab’b second antibody (see Materials and Methods). Stds, molecular weight markers as indicated at the left edge of the figure.

procedure for detecting lower-molecularweight chrc’matin and histone components. Proteins were detected on gels with Coomassie stain and on NC blots with amido black. PPxyproteins were visualized on both NC and ZB blots by the immunochemical procedure which employs horseradish peroxidase-linked goat anti-mouse F(ab’)z second antibody to mouse monoclonal anti-PPxy first antibody.

Figure 2 reveals that immunochemical detection of PPxy-derivatized chromatin is far superior to both Coomassie and amido black staining procedures, being in the range of 10 to 100 times more sensitive. Immunoblot analyses of chromatin fractionated into the H 1 and the non-H1 components are shown in Figs. 3 and 4, respectively. The method is capable of detecting nanogram amounts of

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FIG. 3. SDS-gel electrophoresis and NC blots of the HI histone fraction of rat liver chromatin. (pg) was determined by the Lowry procedure (35). For experimental methods. see Fig. 2.

Protein

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FIG. 4. SDS-gel chromatin. Protein

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electrophoresis and NC blot of the non-HI-containing (pg) was determined by the Lowry procedure (35).

For

histone fraction of rat liver experimental methods, see

Fig. 2.

fractionated histones on NC paper. Figure 5 provides the results of an experiment using ZB as the immobilizing matrix for SDS-gel electrophoresis-resolved chromatin. The sensitivity of the immunochemical detection of resolved chromatin on ZB is approximately IO times that of Coomassie on gels (Fig. 5) but is not as sensitive as immunochemical detection of resolved chromatin on NC (Fig. 2).

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DISCUSSION

PLP has been used to derivatize a large number of diverse proteins such as lactic dehydrogenase ( 14), glucose-&phosphate dehydrogenase (15), fructose- 1,6-bisphosphatase ( 16), aldolase( 17). reovirus transcriptase ( 18), a-thrombin ( 19), ribulose- 1,5-bisphosphate carboxylase/oxygenase (20), diol dehydrase (21), and the recBC enzyme (22). In addition,

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COOMASSIE FIG. 5. SDS-gel electrophoresis as described in Fig. 1.

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IMMUNOBLOT

and ZB blot of rat liver chromatin.

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The conditions

for the ZB blot were

IMMUNOCHEMICAL

DETECTION

it has been used to derivatize Escherichia coli initiation factor 3 (23) and elongation factor G (24). It has been shown to be a useful reagent as well in the study of enzyme complexes (25) hemoglobin function (26) membrane structure (27,28), and steroid receptor function (2932). Thus, in addition to its remarkable versatility as a coenzyme (33,34), PLP is a powerful reagent in its own right and can be applied to many kinds of biochemical analyses. The present study takes advantage of the reactivity of PLP towards t-amino groups of lysine residues in proteins, the ease of covalent linkage of PLP to lysine side chains as the PPxy group using sodium borohydride, and the availability of monoclonal antibodies to the PPxy group. Our procedure is similar in kind to the procedure recently reported by Wojtkowiak et al. (7) and shares with it the following properties: sensitivity, simplicity, and genera1 applicability. It has the following advantages: no organic solvents are required. reactions are carried out efficiently under mild conditions, monoclonal antibodies are essentially reagents in that there is no biological variability as may occur with antisera. and second and third antibodies or proteins can be used at high dilutions ( 1:10.000). In principle. it can be usedwith any matrix that does not react with PLP. It can be used on ZB paper, providing a hitherto unavailable general staining procedure for this protein-immobilizing matrix. In addition, our procedure can ethciently detect low-molecular-weight chromatin and histone proteins (Figs. 2-5) which is probably a consequenceof the fact that these proteins are generally lysine rich. Like the DNP/anti-IDNP procedure of Wojtkowiak et ul. (7), our method depends on amino group derivatization. On a lysine molar equivalent basis,one would expect equal staining intensities.However. as noted (7). one might expect that lower..molecular-weight proteins, on a protein molar equivalent basis, would stain lesswell becausethey would have fewer PLPbinding sites.This appearsto be the casesince. if one compares the five different 75-pg-containing lanes in Fig. 1. one can see that the

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greatest staining densities for the silver, Coomassie, and amido black stains are generally in the area which contain the lowermolecular-weight proteins, whereasthe reverse is true for the corresponding NC and ZB lanes. Thus, the anti-PPxy immunochemical procedure for the detection of proteins will reflect a combination of lysine availability, protein concentration, and protein molecular weight. ACKNOWLEDGMENTS This investigation was supported by PHS Grants CA35878 and AM-32518 from the National Institutes of Health. and by BRSG Grant 2-32908 from the University of Vermont College of Medicine.

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