Direct Immunodetection of Antigens within the Precast Polyacrylamide Gel

Analytical Biochemistry 297, 94 –98 (2001) doi:10.1006/abio.2001.5324, available online at http://www.idealibrary.com on

Direct Immunodetection of Antigens within the Precast Polyacrylamide Gel Surbhi Desai, 1 Boguslawa Dworecki, and Eugene Cichon Pierce Chemical Company, Rockford, Illinois 61105

Received April 6, 2001; published online August 30, 2001

Detection of specific proteins separated by SDS– PAGE is the basis for studying specific antigens. Immunodetection of antigens is commonly performed using Western blotting technique. In this paper we have shown that it is possible to eliminate Western blotting and to detect the antigens directly within the precast polyacrylamide gels by pretreating the gels with 50% isopropanol followed by distilled water treatment. This method would be valuable for large or difficult to transfer proteins. © 2001 Academic Press Key Words: in-gel immunodetection; SDS–PAGE; Western blot; polyacrylamide gels; chemiluminescent substrate; electrophoresis.

Polyacrylamide gel electrophoresis is used to resolve and identify crude and pure protein samples. To determine the complexity of samples, gels are stained for proteins with traditional staining methods. The study of specific enzyme–substrate, antigen–antibody, glycoprotein–lectin, hormone–receptor, or other protein– protein interactions is often necessary. Originally, this type of detection was performed within the gel (1–3). The procedure took several days, as it required lengthy fixing (2–12 h), antibody incubations (up to 12 h), and wash steps (24 –72 h) (4). Proteins were fixed in the gel with trichloroacetic acid or isopropanol and acetic acid. The proteins were detected either with corresponding peroxidase labeled antibody and 3,3-diaminobenzidine or with radiolabeled lectins or antibodies. To reduce the procedure time and to improve signal to noise ratios, detection within the gel was abandoned for Western blotting techniques (5, 6). In Western blotting, proteins are separated in a three-dimensional gel slab and then transferred and immobilized onto a two1 To whom correspondence should be addressed at Pierce Chemical Company, 3747 N. Meridian Road, P.O. Box 117, Rockford, IL 61105.

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dimensional membrane matrix. The membrane is incubated with a specific antibody, which in turn is detected with a “reporter.” Reporters are radiolabeled, fluorescent, or enzyme-labeled molecules that facilitate detection (7). Several factors affect the overall transfer efficiency in Western blotting. These include the electrical current applied, the molecular weight and the net charge of a polypeptide, and the pore size of the polyacrylamide gels. Transfer conditions suited for one polypeptide may not be optimal for another, as the rate of elution depends on its molecular weight. Smaller polypeptides transfer more efficiently than larger ones. The pore size of the membrane may also affect the overall immobilization of polypeptides. In addition, the diffusion of proteins during electrotransfer may affect the bandwidths of the polypeptides immobilized on nitrocellulose (8). In some cases, blotting conditions can destroy relevant epitopes through unfolding of the protein structure upon adsorption to a membrane. In addition to the denaturation that occurs during sample preparation and electrophoresis, renaturation of epitopes is often inefficient and/or incomplete. Because specific antibodies are frequently raised to proteins in their native conformations, the binding of antibodies to blotted proteins is unpredictable (7). Studies have also shown that antigens can be lost from membranes during immunoprocessing (10). These factors suggest that the pattern obtained on a Western blot may not be a true representation of the sample separated by SDS– PAGE. There are also some proteins such as fibrinogen, which do not transfer well to membranes. Other proteins have been reported to bind selectively either to nitrocellulose or PVDF 2 membranes (11). 2

Abbreviations used: GAM-HRP, goat anti-mouse antibody– horseradish peroxidase; GAR-HRP, goat anti-rabbit antibody– horseradish peroxidase; RT, room temperature; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; GST, glutathione S-transferase; IgG, immunoglobulin G; BSA, 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

ELECTROPHORETIC IMMUNODETECION OF ANTIGENS

Detection of antigens within agarose and glyoxyl agarose gels using immunoprobing techniques was introduced due to limitations of Western blotting. Glyoxyl agarose has an advantage over agarose since once the sample is separated by electrophoresis, antigens (via their amine groups) can be fixed in place with sodium cyanoborohydride (NaCNBH 3) (12). Due to the poor molecular sieving properties obtained with the glyoxyl agarose, composite glyoxyl/polyacrylamide gels have been reported as an option. This combined the low permeability feature of polyacrylamide required for molecular sieving with the high permeability feature of agarose required for solid-phase probing. The polyacrylamide was incorporated as a removable filler within the glyoxyl agarose matrix. With the use of agarose or glyoxyl agarose gels, a fixation step for 1–2 h was generally used followed by a wash step. Antibody incubation steps were generally done for 4 –12 h. The antibodies were added either passively by immersing the gel into solution or actively by compressing the gel so it could imbibe the antibody. Unbound antibody was generally removed with the use of a gel dryer or equivalent apparatus. Immunovisualization was performed with the use of various substrates (12). Although polyacrylamide gels are the most widely used means of sample separation, they are not currently used for direct immunoprobing due to the general belief that they are impermeable to antibodies and other large proteins. Solid-phase detection of antigen– antibody, glycoprotein–lectin, hormone–receptor, or other protein–protein interactions directly in gels is valuable as it eliminates the need for the transfer process. With the elimination of the transfer process, there is no need to optimize the transfer conditions and thus no basis for protein denaturation. In addition, the blocking step can be omitted as the antibodies or proteins will not bind nonspecifically to the gels. However, the blocker can be used as a diluent to prevent antibodies from binding nonspecifically to proteins within the gel and as a stabilizer for dilute antibodies. A uniform representation of the separated sample is obtained with in-gel immunostaining since there is no selective transfer of polypeptides. In the present study we have shown that antigens can be detected directly within the precast polyacrylamide gel by pretreating the gel with 50% isopropanol followed by distilled water treatment. Various pure proteins and lysates were separated by SDS–PAGE and the antigens were detected directly or indirectly using HRP-labeled antibodies. In both cases a sensitive chemiluminescent substrate was used. We have also observed that following the detection step, the gel can

bovine serum albumin; PBS, phosphate-buffered saline; ECL, enhanced chemiluminescence; GFP, green fluorescent protein.

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be stripped and reprobed with the same or another antibody pair (data not shown). In addition, we also have successfully stained the gel for total proteins directly after the in-gel immunodetection procedure (Coomassie or zinc stain; data not shown). Even with the zinc stain, no additional gel manipulation (except for 30 min water wash) is required for “reverse” protein staining. Another advantage of the in-gel immunodetection method is that the bands can be detected on both sides of the gel. Using the Western blotting system band intensity is greater on the transfer side of the membrane. MATERIALS AND METHODS

Reagents Pure GST and anti-GST–rabbit polyclonal IgG were obtained from Santa Cruz BioTechnology (Santa Cruz, CA). Escherichia coli bacterial cell lysate expressing recombinant mouse Id-1:GST, Id-2:GST, and Id-3:GST were obtained from B. D. PharMingen (San Diego, CA). Penta His antibody BSA-free mouse was purchased from Qiagen (Chatsworth, CA) and Anti-Living Color A.v. Peptide antibody rabbit was from ClonTech (Palo Alto, CA). Tris– glycine SDS sample buffer (2⫻) from Invitrogen (Novex) (San Diego, CA). ECL Western blotting detection reagent was acquired from Amersham Pharmacia Biotech (Arlington Heights, IL). Developer/ replenisher and fixer/replenisher were obtained from Sigma Chemical Corporation (St. Louis, MO). Precast polyacrylamide gels were purchased from Invitrogen (Novex). StabilZyme–HRP was purchased from SurModics (Eden Prairie, MN). The remaining materials were obtained from Pierce Chemical Company (Rockford, IL): phosphate-buffered saline (PBS); Tween 20; goat anti-rabbit–HRP; goat anti-mouse–HRP; electrophoresis buffer; BlueRanger Prestained Molecular Weight marker mix; 10⫻ Blocker BSA; UnBlot substrate (stable peroxide buffer and luminol enhancer); methanol; ethanol; isopropanol; exposure films; nitrocellulose membrane; and transfer buffer. Sample Preparation and Gel Electrophoresis Pure protein samples were prepared by diluting in 2⫻ sample buffer such that the final protein concentration was 0.1–1 ng/␮l. Lysate samples were diluted 1:10 –1:4000 in 2⫻ sample buffer. All samples were heated at 95°C for 5 min and cooled before loading. The samples were separated by analytical minigel SDS– PAGE systems (Novex). Goat anti-mouse antibody– horseradish peroxidase (GAM-HRP) or goat anti-rabbit antibody– horseradish peroxidase (GAR-HRP) were reconstituted at 1mg/ml in Milli-Q water and diluted to 10 ␮g/ml in StabilZyme–HRP stabilizing solution.

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FIG. 1. Comparison of alcohol chain length vs detection limits. Pure GFP/6⫻His-tagged proteins were separated by SDS–PAGE (Novex 10 – 20% Tris– glycine gels). One gel was pretreated with Milli-Q water (control gel A), one with 50% methanol (B), one with 50% ethanol (C), and one with 50% isopropanol (D). All gels were processed for in-gel immunodetection with a 1:500 dilution of mouse anti-penta-His antibody followed by the HRP-labeled secondary antibody. Lanes 1–5, 10, 5, 2.5, 1.25, and 0.5 ng pure GFP/6⫻His-tagged, respectively.

Pretreatment and “In-Gel Immunodetection” Unless otherwise stated, the gels were pretreated for 15 min with 50 ml of 50% isopropanol/water and then washed for 15 min with 100 ml water (Milli-Q). The primary antibody was diluted in 1⫻ Blocker BSA/PBS/ 0.05% Tween 20. The gels were incubated in 20 ml of diluted primary antibody with mild agitation for 1 h at room temperature (RT). The gels were washed 3 ⫻ 10 min with 100 ml PBS/0.05% Tween 20. The HRP-labeled secondary antibody (GAM-HRP or GAR-HRP) solution at 10 ␮g/ml stock was diluted 1:250 or 1:500 in 1⫻ Blocker BSA/PBS/0.05% Tween 20. The appropriate diluted antibody solution (20 ml) was added to each gel. The gels were incubated with mild agitation for 1 h at RT and then washed 3 ⫻ 10 min with 100 ml PBS/ 0.05% Tween 20. The gels were incubated for 5 min at RT in 10 ml unblot substrate working reagent, which was prepared by mixing 5 ml of unblot stable peroxide buffer with 5 ml of unblot luminol enhancer. As a final step, the gels were washed for 15 s with Milli-Q water. Using the handling tool (e.g., gel knife), the gels were placed between cellophane sheets and exposed to X-ray film for 30 s, 1 min, or 5 min. The films were developed for 1 min in developer/replenisher solution, rinsed in water, fixed for 1 min in fixer/replenisher solution, and again rinsed with water. When a directly labeled HRP primary antibody was used, the secondary antibody HRP incubation step and the following wash step were omitted. The remaining steps were as previously described. Immunoblotting Procedure After electrophoresis, the gels were transferred to nitrocellulose membranes using a Bio-Rad minigel transfer

unit at 200 mA for 2 h. The membranes were blocked overnight in 1⫻ Blocker BSA/PBS/0.05% Tween 20. The following day the membranes were incubated with mild agitation in 20 ml of primary antibody diluted in 1⫻ Blocker BSA/PBS/0.05% Tween 20. The membranes were incubated for 1 h at RT and then washed 3 ⫻ 10 min with 100 ml PBS/0.05% Tween 20. The HRP-labeled secondary antibody (GAM-HRP or GAR-HRP) solution at 10 ␮g/ml stock was diluted 1:250 to 1:500 in 1⫻ Blocker BSA/PBS/0.05% Tween 20. The appropriate diluted antibody solution (20 ml) was added to each membrane. The membranes were incubated with agitation for 1 h at RT and then washed 3 ⫻ 10 min with 100 ml PBS/0.05% Tween 20. The membranes were incubated for 5 min at RT in 10 ml ECL substrate working reagent, which was prepared by mixing 5 ml of detection reagent 1 with 5 ml of detection reagent 2. The membranes were placed between cellophane sheets and exposed to X-ray film for 30 s, 1 min, or 5 min. The films were developed for 1 min in developer/replenisher solution, rinsed in water, fixed for 1 min in fixer/replenisher solution, and again rinsed with water. RESULTS AND DISCUSSION

With our novel in-gel immunodetection technique combined with an extremely sensitive chemiluminescent substrate we have shown that it is possible to detect antigens within the precast gel. Pretreatment of the gels with an alcohol is essential for the detection. We have compared methanol, ethanol, and isopropanol and found a relationship between the alcohol chain length and the detection sensitivity (Fig. 1). Isopropanol has the longer carbon chain length and caused the highest degree of gel shrinkage (Table 1). Following

TABLE 1

Comparison of Alcohol Chain Length and the Degree of Gel Shrinkage Pretreatment solution

Area % decrease following pretreatment step

Area % increase following rehydration

Water 50% MeOH 50% EtOH 50% IPA

0 ⫺12.53 ⫺16.91 ⫺25.59

⫹16.19 ⫹15.14 ⫹14.86 ⫹11.33

Note. Following SDS–PAGE, four 10 –20% Novex gels were removed from the cassettes, placed on the protective sheet, and photocopied. Next, one gel was placed in 50 ml of 50% methanol (B), 50% ethanol (C), or 50% isopropanol (D) and pretreated for 15 min at room temperature with mild agitation. Control gel (A) was left in the protective sheet for 15 min. After the pretreatment step, the gels were placed on the plastic protective sheet and photocopied. Then, all gels (including the control gel) were rehydrated for 15 min in 100 ml of Milli-Q water. Following rehydration, all gels were placed on the plastic protective sheet and photocopied to obtain final measurements.

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body detected only the GFP/6⫻His-tagged proteins. Therefore, as with the Western blotting system, in-gel immunodetection illustrates specific binding of antibody to the corresponding antigens. The actual sensitivities obtained by Western blotting and in-gel immunodetection are also demonstrated in Fig. 2. In this experiment, E. coli lysate expressing GFP/6⫻His-tagged protein was serially diluted and applied to 10 –20% Tris– glycine Novex gels. The gel and Western blot were probed with identical dilutions FIG. 2. Comparison of sensitivity between in-gel immunodetection and Western blotting using mouse anti-penta-His antibody. E. coli bacterial lysate expressing GFP/6⫻His-tagged were separated by SDS–PAGE on Novex 10 –20% Tris– glycine gels. (A) One gel was processed for in-gel immunodetection with a 1:500 dilution of antipenta-His monoclonal mouse followed by goat anti-mouse–HRP-conjugated antibody (1:250 dilution). (B) A second gel was transferred to nitrocellulose membrane using the Bio-Rad minigel transfer unit and then membrane was processed with identical antibody dilutions as described for in-gel immunodetection. Lanes 1–5, E. coli bacterial GFP/6⫻His-tagged lysate diluted 1:100, 1:250, 1:1000, 1:2000, and 1:4000, respectively.

rehydration, all the gels were found to be slightly larger than the original size, irrespective of the alcohol used. We believe that the alcohol pretreatment changes the nature of the polyacrylamide gel making it more permeable to antibodies/proteins thus increasing the sensitivity and detection of in-gel immunodetection technique. To show versatility of the system, different antigen– antibody pairs were tested. Pure proteins, crude yeast, and bacterial lysates were separated by SDS–PAGE and detected using corresponding antibody systems. The in-gel immunodetection system was successfully tested with various polyacrylamide gradient and nongradient (1.0 –1.5 mm thick) precast Tris– glycine, Bis– Tris, Tris–tricine, and Tris–acetate gels from Novex, Tris– glycine Criterion gels from Bio-Rad and Tris– glycine BioWhittaker gels. Bio-Rad Ready Gels, I-Gels, and Zaxis gels displayed a 25-fold lower sensitivity and/or higher backgrounds (data not shown). The gel thickness affects detection sensitivity. Thinner gels (1.0 vs 1.5 mm) need shorter exposure time to film or a lower dilution of the antibody compared to thicker gels. This suggests that the antibodies bind antigens more readily in the thinner gels since the protein is closer to the periphery. We have shown selectivity of in-gel immunodetection system by separating GFP/6⫻His-tagged and nontagged GFP proteins on two identical gels. One gel was probed with a polyclonal antibody specific for GFP and the other with a monoclonal antibody specific for Histagged proteins. The results demonstrate (data not shown) selectivity since the Anti-Living Color A.v. Peptide identified the GFP bands, and the penta-His anti-

FIG. 3. Comparison of in-gel immunodetection with Western blotting using rabbit anti-GST antibody. Pure GST protein, bacterial GST lysate: Id1, Id2, and Id3 and yeast GST lysates were separated by SDS–PAGE (Novex 4 –20% Tris– glycine gels). (A) One gel was processed for in-gel immunodetection with a 1:1000 dilution of antiGST rabbit followed by a 1:500 dilution of HRP-labeled goat antirabbit antibody. (B) A second gel was transferred to nitrocellulose membrane using the Bio-Rad mini gel transfer unit and the membrane was processed with identical antibody dilutions as described for in-gel immunodetection. (A) Lanes 1–3, 10, 5, and 1 ng pure GST, respectively; lanes 5 and 6, bacterial Id3 and Id2 lysates diluted 1:50; lanes 7–9, bacterial lysate Id1 diluted 1:50, 1:20, and 1:100, respectively; Lanes 11 and 12, yeast GST lysate 1:20 and 1:100, respectively. No sample was applied to lanes 4 and 10. (B) Western blot. Lanes 1–3, 10, 5, and 1 ng pure GST, respectively; lanes 4 and 5, yeast GST lysate 1:20 and 1:100, respectively; lanes 6 and 7, bacterial Id3 and Id2 lysates diluted 1:50; lanes 8 –10, bacterial lysate Id1 diluted 1:20, 1:50, and 1:100, respectively.

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of mouse anti-penta-His antibody followed by secondary HRP-conjugated antibody. Both systems showed detection down to 1:2000 dilution of the lysate. However, it should be realized that the antibody dilutions best suited for one system are not always optimal for another. The two techniques may also require different exposure times for optimal results. In a direct comparison of in-gel immunodetection to Western blotting of GST (pure and crude), we obtained equivalent results using both techniques (Fig. 3). Pure GST was detected down to 1 ng. With the direct gel detection, results are obtained in a shorter time (3 to 14 h less). In addition, the background and nonspecific binding is reduced. This is because with Western blotting, nonspecific binding of any reagent can result in binding of additional reagents in the subsequent steps. In this analysis, comparisons between Western blot and in-gel immunodetection systems suggest uneven transfer of proteins. In the Western blot, the higher molecular weight GST aggregate bands are not apparent as in the in-gel immunodetection due to inefficient transfer of higher molecular weight proteins. The Western blot (Fig. 3B) has some loss in signal in lane 1 as a result of inefficient protein transfer caused by an air bubble trapped between the gel and the membrane. The in-gel immunodetection system showed compatibility with directly labeled and indirectly labeled antibodies. The directly labeled antibody may show more intense bands (data not shown) suggesting that the HRP labeled secondary antibody has tendencies to bind bands nonspecifically. Directly labeled primary antibody may be used to reduce nonspecific binding caused by HRP-labeled secondary antibody. Our studies prove that it is possible to detect antigens directly within the precast gel by pretreating the gel with 50% isopropanol followed by distilled water treatment. Comparisons with Western blotting show that sensitivities within 1 order of magnitude can be obtained with the in-gel immunodetection technique.

In addition, the in-gel immunodetection system demonstrated greater specificity. GFP samples (6⫻Histagged or nontagged) separated by SDS–PAGE and probed with two different antibodies successfully detected specific antigens. REFERENCES 1. Burridge, K. (1976) Changes in cellular glycoproteins after transformations: Identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Proc. Natl. Acad. Sci. USA 73, 4457– 4461. 2. Rosta, J. A., Kelly, P. T., and Cotman, C. W. (1977) The identification of membrane glyco components in polyacrylamide gels: A rapid method using 125-I labeled lectins. Anal. Biochem. 80, 366 – 372. 3. Olden, K., and Yamada, K. M. (1977) Direct detection of antigens in sodium dodecyl sulfate–polyacrylamide gels. Anal. Biochem. 78, 483– 490. 4. Adair, W. S., Jurivich, D., and Goodenough, U. S. (1978) Localization of cellular antigens in sodium dodecyl sulfate polyacrylamide gels. J. Cell Biol. 78, 281–285. 5. Towbin, H., Staehlin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350 – 4354. 6. Renart, J., Reiser, J., and Stark, G. R. (1979) Transfer from proteins from gels to diazobenzyloxymethyl paper and detection with antisera: A method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA 76, 3116 –3120. 7. Gersten, D. M. (1996) Gel Electrophoresis: Proteins (Rickwood, D., Ed.), Wiley, New York. 8. Lin, W., and Kasamatsu, H. (1983) On electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal. Biochem. 128, 302–311. 9. Deleted in proof. 10. DenHollander, N., and Befus, D. (1989) Loss of antigens from immunoblotting membranes. J. Immunol. Methods 122, 129 – 135. 11. Reddy, V. M., and Kumar, B. (2000) Interactions of Mycobacterium avium complex with human respiratory epithelial cells. 12. Shainoff, J. R. (1993) Electrophoresis and direct immunodetection on glyoxal agarose and polyacrylamide composites. Adv. Electrophoresis 6, 64 –177.