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Double Immunodetection of Proteins Transferred onto a Membrane Using Two Different Chemiluminescent Reagents
NOTES & TIPS Double Immunodetection of Proteins Transferred onto a Membrane Using Two Different Chemiluminescent Reagents Shunji Hattori1 and Hitomi Fujisaki Nippi Research Institute of Biomatrix, 1-1-1 Senjumidoricho, Adachi-ku, Tokyo, 120 Japan Received February 12, 1996
Recently luminogenic substrates have been used as sensitive and nonradioactive immunological tools for the detection of proteins in immunoblot assays (1–3). We have developed a simple method for the double immunodetection of different proteins on the same transfer membrane using different kinds of luminogenic substrates that are activated with either peroxidase or alkaline phosphatase. In this double immunodetection method, two different antibodies, each derived from different species, were mixed and used together as the first antibodies. Then a mixed solution of peroxidase and alkaline phosphatase conjugates of anti-IgG antibodies, corresponding to the species used, was employed as the second antibodies. The transfer membrane was reacted first with the substrate of the peroxidase (ECL, Amersham). Subsequently, the membrane was reacted with the substrate of alkaline phosphatase (CSPD, Tropix Inc., MA) in the presence of sodium azide. Using this protocol, we achieved an independent immunological detection of different proteins. Material and Methods Antigens. Fibronectin from bovine plasma was purified by gelatin–Sepharose affinity chromatography (4). An electrophoresis calibration kit containing phosphorylase b, ovalbumin, and bovine serum albumin (BSA)2 was purchased from Pharmacia. Human foreskin keratinocytes (HFK) were grown in K110 medium (containing 30 mM Ca2/, Kyokuto Pharmaceutical Co.) supplemented with bovine pituitary extract. HFK (pas1
To whom correspondence should be addressed. Fax: /81-338709631. 2 Abbreviations used: BSA, bovine serum albumin; Coomassie R, Coomassie brilliant blue R-250; HFK, human foreskin keratinocytes; PBS, phosphate-buffered saline; SDS–PAGE, SDS–polyacrylamide gel electrophoresis. ANALYTICAL BIOCHEMISTRY ARTICLE NO. 0517
sage 4) cultured for 3 days in K110 medium or in the presence of CaCl2 (1 mM) were extracted with 8 M urea in phosphate-buffered saline (PBS). The protein content of each extract was measured using the Bio-Rad protein assay reagent (Bio-Rad Laboratories). Antibody. Murine monoclonal anti-fibronectin antibody (FC113) was prepared. This monoclonal antibody recognizes the C-terminal heparin-binding domain of fibronectin. Murine monoclonal anti-b-actin antibody (A-4700) was purchased from Sigma. Rabbit polyclonal anti-human keratin antibody was from Medac Gesellschaft fu¨r Klimische Spezialpreparate MBH (Germany). Rabbit polyclonal anti-ovalbumin antibody, peroxidase-conjugated goat anti-rabbit (or mouse) IgG antibody, and alkaline phosphatase-conjugated goat anti-mouse (or rabbit) IgG antibody were from Organon Teknika Co. Double immunodetection of proteins. Proteins separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) were transferred to a PVDF membrane (Immobilon-P, Millipore Co.) in 0.05 M borate buffer, pH 9.5. The membrane was blocked with PBS containing 0.05% Triton X-100–1% BSA and incubated with a mixture of murine monoclonal antibodies and rabbit polyclonal antibodies for 2 h at room temperature. After the membrane was washed with PBS containing 0.05% Triton X-100, it was incubated with a mixture of peroxidase-conjugated goat anti-rabbit (or mouse) IgG antibody (1/1000 dilution) and alkaline phosphatase-conjugated goat anti-mouse (or rabbit) IgG antibody (1/1000 dilution). After the washes (3 1 5 min), the PVDF membrane was incubated in the ECL reagent; the membrane was wrapped in plastic and exposed to HR-S X-ray film (Fuji Photo Film Co., Ltd.) for 1 min. The membrane was then unwrapped and washed twice (5 min) with alkaline phosphatase assay buffer (0.1 M diethanolamine, 1 mM MgCl2 , pH 10.0) containing 0.02% sodium azide (to inhibit residual peroxidase activity), and the diminished chemiluminescence compared with the previous assay was evaluated by exposing the membrane to the X-ray film. Next, the membrane was incubated in the alkaline phosphatase chemiluminescent substrate (50 ml of 0.24 mM CSPD in 5 ml of assay buffer) containing 1:20 Nitro Block (Tropix Inc.) for 5 min and the membrane was exposed to X-ray film for 1–10 min. Exposure time was altered depending on the intensity of the signals.
243, 277–279 (1996)
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FIG. 1. Double immunodetection of fibronectin and ovalbumin. The mixture of fibronectin (FN), phosphorylase b (Pho b), bovine serum albumin (BSA), and ovalbumin (OVA) was separated by 8% SDS– PAGE and stained with Coomassie R (lanes 1–4). The proteins transferred on the PVDF membrane were incubated with mixture of murine monoclonal anti-FN antibody and rabbit polyclonal anti-OVA antibody (1/1000 dilution). First, FN was detected with peroxidaseconjugated anti-mouse IgG using ECL reagent (anti FN-P, lanes 5– 7). After a wash with diethanolamine buffer (pH 10.0) containing sodium azide (0.02%), OVA was detected with alkaline phosphataseconjugated anti-rabbit IgG using CSPD (anti-OVA-AP) on the same membrane (lanes 8–10). Direct detection of OVA was also shown (lanes 11–13). Amount of each protein applied on the SDS–PAGE was 1 mg (lane 1), 100 ng (lanes 2, 5, 8, and 11), 10 ng (lanes 3, 6, 9, and 12), or 1 ng (lanes 4, 7, 10, and 13).
ity. The doublet bands that were recognized by antiovalbumin antibody probably correspond to ovalbumin with different degrees of phosphorylation. Next, this method was applied to the detection of proteins from cultured keratinocytes. It is known that the addition of CaCl2 induces the differentiation of HFK (5), and that the expression of the keratin subtypes shift from the basal cell types keratin 5 (58 kDa) and keratin 14 (50 kDa) to the stratified cell types keratin 1 (68 kDa) and keratin 10 (56.5 kDa) (6, 7). Using this culture system, we chose keratin and actin as the antigens for testing the double immunodetection technique. Proteins detected by Coomassie R did not show prominent differences among the HFK extracts from the different culture conditions (Fig. 2, lanes 2–5). With the first exposure by the ECL reagent, the chemiluminescence of the keratin bands including keratins 5, 14, and 19 (Fig. 2, lane 6) was detected. In the extract from CaCl2-treated HFK, keratins 1 and 10 were detected in addition to the keratins detected in lane 5 (Fig. 2, lane 7). The second exposure, using CSPD, allowed the detection of the actin band (42 kDa) (Fig. 2, lanes 8 and 9), without display of any keratin bands. Actin could easily be distinguished from keratin 19 (40 kDa) despite their very similar molecular weights. Even when the order of detection was reversed, actin
Results and Discussion First, a mixture of phosphorylase b, ovalbumin, BSA, and fibronectin was used as a model sample for the double immunodetection method. Various concentrations of these proteins were separated by 8% SDS– PAGE and were stained with Coomassie brilliant blue R-250 (Coomassie R) (Fig. 1, lanes 1–4) and protein blots were prepared. To detect fibronectin and ovalbumin, murine monoclonal anti-fibronectin antibody and rabbit polyclonal anti-ovalbumin antibody were used as the primary probes and peroxidase-conjugated anti-mouse IgG antibody and alkaline phosphataseconjugated anti-rabbit IgG antibody were used as the second probes. First, the fibronectin band was detected by the anti-fibronectin antibody using ECL reagent (Fig. 1, lanes 5–7); 100 ng was found to produce a strong signal. Then after a wash with the alkaline phosphatase assay buffer (pH 10.0) containing 0.02% sodium azide, the presence of ovalbumin was assayed; as little as 1 ng was detected with CSPD and no chemiluminescence from the fibronectin band was found (Fig. 1, lanes 8–10). Because direct detection of ovalbumin (without prior use of the peroxidase-conjugated antibody and ECL exposure) showed a similar staining pattern (Fig. 1, lanes 11–13), the double immunodetection method did not appear to affect the detection sensitiv-
FIG. 2. Detection of actin and keratins from keratinocytes. Extracts from keratinocytes cultured in K110 medium (lanes 2, 3, 6, 8, 10, and 12) or in the presence of CaCl2 (1 mM) (lanes 4, 5, 7, 9, 11, and 13) were separated by 10% gel SDS–PAGE and stained with Coomassie R (lanes 2–5). Lane 1 shows prestained protein standard (Bio-Rad Laboratories) and left margin shows molecular weight of the standard proteins. The proteins transferred on the membrane were incubated with mixture of murine monoclonal anti-actin IgG (1/1000 dilution) and rabbit polyclonal anti-keratin IgG (1/2000 dilution), and then membrane was reacted with mixture of peroxidaseconjugated anti-rabbit IgG and alkaline phosphatase-conjugated anti-mouse IgG antibody. First, keratins were detected with peroxidase substrate (lanes 6 and 7); subsequently, actin was detected with alkaline phosphatase substrate on the same membrane (lanes 8 and 9). Immunodetection performed in the reverse order was shown in lanes 10 and 11 for actin with peroxidase substrate and lanes 12 and 13 for keratins with alkaline phosphatase substrate. The amount of proteins applied on the SDS–PAGE was 5 mg (lanes 2 and 4) or 500 ng (lanes 3, 5 and 6–13).
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first (Fig. 2, lanes 10 and 11) followed by keratin detection (Fig. 2, lanes 12 and 13), the same results as previously were obtained. Quantitative analysis of the keratin as influenced by the CaCl2 treatment could be accomplished using the actin signal as an internal standard on the same membrane. Once an appropriate set of antibodies generated from different species was chosen, this highly sensitive double immunodetection on the same transfer membrane method has the following benefits. (1) A correct comparison of band positions (especially protein spots on twodimensional electrophoresis) can be made. (2) The number of electrophoresis runs can be reduced and valuable samples can thus be conserved. (3) This method should be more quantitative than other double immunodetection methods which use SDS to ‘‘clean’’ the blot before the second detection is likely therefore to remove some of the antigens (8). Such an SDS step was not required in our method. REFERENCES 1. Laing, P. (1986) Anal. Biochem. 92, 161–165. 2. Meier, T., Arni, S., Malarkannan, S., Poincelet, M., and Hoessli, D. (1992) Anal. Biochem. 204, 220–226. 3. Gillespie, P. G., and Hudspeth, A. J. (1991) Proc. Natl. Acad. Sci. USA 88, 2563–2567. 4. Sakai, K., Fujii, T., and Hayashi, T. (1994) J. Biochem. 115, 415– 421. 5. Yuspa, S. H., Kilkenny, A. E., Steinert, P. M., and Roop, D. R. (1989) J. Cell Biol. 109, 1207–1217. 6. Fuchs, E., and Green, H. (1980) Cell 19, 1033–1042. 7. Lane, E. B. (1993) in Connective Tissue and its Heritable Disorders (Royce, P. M., and Steinmann, B., Eds.), pp. 237–247, Wiley-Liss, New York. 8. Li, L., Tennenbaum, T., and Yuspa, S. H. (1996) J. Invest. Dermatol. 106, 254–260.
Thin-Layer Affinity Chromatography in Analysis of Protein–Ligand Affinity Mika P. A. Laitinen,1 Kirsi M. Sojakka, and Matti Vuento Department of Biological and Environmental Science, University of Jyva¨skyla¨, Vapaudenkatu 4, FIN-40100 Jyva¨skyla¨, Finland Received June 19, 1996
Work with proteins often involves operations that can induce conformational changes and affect the bind1
To whom correspondence should be addressed. Fax: /358 (0)14 602 221. ANALYTICAL BIOCHEMISTRY ARTICLE NO.
ing of protein to its ligand. Hence, it is important to be able to monitor the affinity state of the protein studied relative to the original native state. Quantitative methods for assessing affinity parameters involve setting up equilibrium mixtures of protein and ligand and analyzing the concentrations of bound and free species by various physicochemical methods (1). From such measurements, the dissociation constant of the binding reaction can usually be derived. Unfortunately, these methods are tedious and do not allow a rapid check to be made on the affinity state of the protein. Affinity chromatography has been previously used for the estimation of dissociation constants (2, 3). Recent developments in affinity chromatography have introduced thin-layer techniques in the rapid analysis of antigen binding to antibody (4–7), analogous to protein–ligand interaction. In particular, Lou et al. (5) used several discrete antibody zones immobilized on membrane to analyze lipoprotein concentration in serum. That assay was based on a competition of lipoprotein present in a sample with lipoprotein labeled with colloidal selenium for binding to the antibody zones. These developments led us to test the use of thin-layer affinity chromatography in studying the affinity of protein–ligand interaction after perturbation of protein by denaturation. The method was used in determination of the transition temperature of heat-denatured fibronectin and heatdenatured succinylated avidin on the basis of their binding to polyclonal antibody and to biotin, respectively. On a strip of nitrocellulose membrane, a capture region comprising eight discrete capture zones was set up by allowing adsorption of the ligand or protein-conjugated ligand onto the membrane. The protein in its reference affinity state was labeled with colloidal gold, and the labeled conjugate was allowed to comigrate with nonlabeled protein through the capture region. During migration, the labeled and nonlabeled protein competed for binding to the ligand. The degree to which the discrete capture zones bound the labeled reference protein was dependent on the affinity state of the studied protein. Materials and Methods Nitrocellulose membrane of a nominal pore size of 12 mm was obtained from Sleicher and Schuell (Darmstadt, Germany). Succinic anhydride, ovalbumin, Sacetylmercaptosuccinic anhydride, N-succinimidyl-4( N - maleimidomethyl ) - cyclohexane - 1 - carboxylate (SMCC),2 and biotinamidocaproate N-hydroxysuccinimide ester were obtained from Sigma Chemical Co. (St. 2 Abbreviations used: SMCC, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate; HABA, 4*-hydroxyazobenzene-2carboxylic acid.
243, 279–282 (1996)
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Recently luminogenic substrates have been used as sensitive and nonradioactive immunological tools for the detection of proteins in immunoblot assays (1–3). We have developed a simple method for the double immunodetection of different proteins on the same transfer membrane using different kinds of luminogenic substrates that are activated with either peroxidase or alkaline phosphatase. In this double immunodetection method, two different antibodies, each derived from different species, were mixed and used together as the first antibodies. Then a mixed solution of peroxidase and alkaline phosphatase conjugates of anti-IgG antibodies, corresponding to the species used, was employed as the second antibodies. The transfer membrane was reacted first with the substrate of the peroxidase (ECL, Amersham). Subsequently, the membrane was reacted with the substrate of alkaline phosphatase (CSPD, Tropix Inc., MA) in the presence of sodium azide. Using this protocol, we achieved an independent immunological detection of different proteins. Material and Methods Antigens. Fibronectin from bovine plasma was purified by gelatin–Sepharose affinity chromatography (4). An electrophoresis calibration kit containing phosphorylase b, ovalbumin, and bovine serum albumin (BSA)2 was purchased from Pharmacia. Human foreskin keratinocytes (HFK) were grown in K110 medium (containing 30 mM Ca2/, Kyokuto Pharmaceutical Co.) supplemented with bovine pituitary extract. HFK (pas1
To whom correspondence should be addressed. Fax: /81-338709631. 2 Abbreviations used: BSA, bovine serum albumin; Coomassie R, Coomassie brilliant blue R-250; HFK, human foreskin keratinocytes; PBS, phosphate-buffered saline; SDS–PAGE, SDS–polyacrylamide gel electrophoresis. ANALYTICAL BIOCHEMISTRY ARTICLE NO. 0517
sage 4) cultured for 3 days in K110 medium or in the presence of CaCl2 (1 mM) were extracted with 8 M urea in phosphate-buffered saline (PBS). The protein content of each extract was measured using the Bio-Rad protein assay reagent (Bio-Rad Laboratories). Antibody. Murine monoclonal anti-fibronectin antibody (FC113) was prepared. This monoclonal antibody recognizes the C-terminal heparin-binding domain of fibronectin. Murine monoclonal anti-b-actin antibody (A-4700) was purchased from Sigma. Rabbit polyclonal anti-human keratin antibody was from Medac Gesellschaft fu¨r Klimische Spezialpreparate MBH (Germany). Rabbit polyclonal anti-ovalbumin antibody, peroxidase-conjugated goat anti-rabbit (or mouse) IgG antibody, and alkaline phosphatase-conjugated goat anti-mouse (or rabbit) IgG antibody were from Organon Teknika Co. Double immunodetection of proteins. Proteins separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) were transferred to a PVDF membrane (Immobilon-P, Millipore Co.) in 0.05 M borate buffer, pH 9.5. The membrane was blocked with PBS containing 0.05% Triton X-100–1% BSA and incubated with a mixture of murine monoclonal antibodies and rabbit polyclonal antibodies for 2 h at room temperature. After the membrane was washed with PBS containing 0.05% Triton X-100, it was incubated with a mixture of peroxidase-conjugated goat anti-rabbit (or mouse) IgG antibody (1/1000 dilution) and alkaline phosphatase-conjugated goat anti-mouse (or rabbit) IgG antibody (1/1000 dilution). After the washes (3 1 5 min), the PVDF membrane was incubated in the ECL reagent; the membrane was wrapped in plastic and exposed to HR-S X-ray film (Fuji Photo Film Co., Ltd.) for 1 min. The membrane was then unwrapped and washed twice (5 min) with alkaline phosphatase assay buffer (0.1 M diethanolamine, 1 mM MgCl2 , pH 10.0) containing 0.02% sodium azide (to inhibit residual peroxidase activity), and the diminished chemiluminescence compared with the previous assay was evaluated by exposing the membrane to the X-ray film. Next, the membrane was incubated in the alkaline phosphatase chemiluminescent substrate (50 ml of 0.24 mM CSPD in 5 ml of assay buffer) containing 1:20 Nitro Block (Tropix Inc.) for 5 min and the membrane was exposed to X-ray film for 1–10 min. Exposure time was altered depending on the intensity of the signals.
243, 277–279 (1996)
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FIG. 1. Double immunodetection of fibronectin and ovalbumin. The mixture of fibronectin (FN), phosphorylase b (Pho b), bovine serum albumin (BSA), and ovalbumin (OVA) was separated by 8% SDS– PAGE and stained with Coomassie R (lanes 1–4). The proteins transferred on the PVDF membrane were incubated with mixture of murine monoclonal anti-FN antibody and rabbit polyclonal anti-OVA antibody (1/1000 dilution). First, FN was detected with peroxidaseconjugated anti-mouse IgG using ECL reagent (anti FN-P, lanes 5– 7). After a wash with diethanolamine buffer (pH 10.0) containing sodium azide (0.02%), OVA was detected with alkaline phosphataseconjugated anti-rabbit IgG using CSPD (anti-OVA-AP) on the same membrane (lanes 8–10). Direct detection of OVA was also shown (lanes 11–13). Amount of each protein applied on the SDS–PAGE was 1 mg (lane 1), 100 ng (lanes 2, 5, 8, and 11), 10 ng (lanes 3, 6, 9, and 12), or 1 ng (lanes 4, 7, 10, and 13).
ity. The doublet bands that were recognized by antiovalbumin antibody probably correspond to ovalbumin with different degrees of phosphorylation. Next, this method was applied to the detection of proteins from cultured keratinocytes. It is known that the addition of CaCl2 induces the differentiation of HFK (5), and that the expression of the keratin subtypes shift from the basal cell types keratin 5 (58 kDa) and keratin 14 (50 kDa) to the stratified cell types keratin 1 (68 kDa) and keratin 10 (56.5 kDa) (6, 7). Using this culture system, we chose keratin and actin as the antigens for testing the double immunodetection technique. Proteins detected by Coomassie R did not show prominent differences among the HFK extracts from the different culture conditions (Fig. 2, lanes 2–5). With the first exposure by the ECL reagent, the chemiluminescence of the keratin bands including keratins 5, 14, and 19 (Fig. 2, lane 6) was detected. In the extract from CaCl2-treated HFK, keratins 1 and 10 were detected in addition to the keratins detected in lane 5 (Fig. 2, lane 7). The second exposure, using CSPD, allowed the detection of the actin band (42 kDa) (Fig. 2, lanes 8 and 9), without display of any keratin bands. Actin could easily be distinguished from keratin 19 (40 kDa) despite their very similar molecular weights. Even when the order of detection was reversed, actin
Results and Discussion First, a mixture of phosphorylase b, ovalbumin, BSA, and fibronectin was used as a model sample for the double immunodetection method. Various concentrations of these proteins were separated by 8% SDS– PAGE and were stained with Coomassie brilliant blue R-250 (Coomassie R) (Fig. 1, lanes 1–4) and protein blots were prepared. To detect fibronectin and ovalbumin, murine monoclonal anti-fibronectin antibody and rabbit polyclonal anti-ovalbumin antibody were used as the primary probes and peroxidase-conjugated anti-mouse IgG antibody and alkaline phosphataseconjugated anti-rabbit IgG antibody were used as the second probes. First, the fibronectin band was detected by the anti-fibronectin antibody using ECL reagent (Fig. 1, lanes 5–7); 100 ng was found to produce a strong signal. Then after a wash with the alkaline phosphatase assay buffer (pH 10.0) containing 0.02% sodium azide, the presence of ovalbumin was assayed; as little as 1 ng was detected with CSPD and no chemiluminescence from the fibronectin band was found (Fig. 1, lanes 8–10). Because direct detection of ovalbumin (without prior use of the peroxidase-conjugated antibody and ECL exposure) showed a similar staining pattern (Fig. 1, lanes 11–13), the double immunodetection method did not appear to affect the detection sensitiv-
FIG. 2. Detection of actin and keratins from keratinocytes. Extracts from keratinocytes cultured in K110 medium (lanes 2, 3, 6, 8, 10, and 12) or in the presence of CaCl2 (1 mM) (lanes 4, 5, 7, 9, 11, and 13) were separated by 10% gel SDS–PAGE and stained with Coomassie R (lanes 2–5). Lane 1 shows prestained protein standard (Bio-Rad Laboratories) and left margin shows molecular weight of the standard proteins. The proteins transferred on the membrane were incubated with mixture of murine monoclonal anti-actin IgG (1/1000 dilution) and rabbit polyclonal anti-keratin IgG (1/2000 dilution), and then membrane was reacted with mixture of peroxidaseconjugated anti-rabbit IgG and alkaline phosphatase-conjugated anti-mouse IgG antibody. First, keratins were detected with peroxidase substrate (lanes 6 and 7); subsequently, actin was detected with alkaline phosphatase substrate on the same membrane (lanes 8 and 9). Immunodetection performed in the reverse order was shown in lanes 10 and 11 for actin with peroxidase substrate and lanes 12 and 13 for keratins with alkaline phosphatase substrate. The amount of proteins applied on the SDS–PAGE was 5 mg (lanes 2 and 4) or 500 ng (lanes 3, 5 and 6–13).
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first (Fig. 2, lanes 10 and 11) followed by keratin detection (Fig. 2, lanes 12 and 13), the same results as previously were obtained. Quantitative analysis of the keratin as influenced by the CaCl2 treatment could be accomplished using the actin signal as an internal standard on the same membrane. Once an appropriate set of antibodies generated from different species was chosen, this highly sensitive double immunodetection on the same transfer membrane method has the following benefits. (1) A correct comparison of band positions (especially protein spots on twodimensional electrophoresis) can be made. (2) The number of electrophoresis runs can be reduced and valuable samples can thus be conserved. (3) This method should be more quantitative than other double immunodetection methods which use SDS to ‘‘clean’’ the blot before the second detection is likely therefore to remove some of the antigens (8). Such an SDS step was not required in our method. REFERENCES 1. Laing, P. (1986) Anal. Biochem. 92, 161–165. 2. Meier, T., Arni, S., Malarkannan, S., Poincelet, M., and Hoessli, D. (1992) Anal. Biochem. 204, 220–226. 3. Gillespie, P. G., and Hudspeth, A. J. (1991) Proc. Natl. Acad. Sci. USA 88, 2563–2567. 4. Sakai, K., Fujii, T., and Hayashi, T. (1994) J. Biochem. 115, 415– 421. 5. Yuspa, S. H., Kilkenny, A. E., Steinert, P. M., and Roop, D. R. (1989) J. Cell Biol. 109, 1207–1217. 6. Fuchs, E., and Green, H. (1980) Cell 19, 1033–1042. 7. Lane, E. B. (1993) in Connective Tissue and its Heritable Disorders (Royce, P. M., and Steinmann, B., Eds.), pp. 237–247, Wiley-Liss, New York. 8. Li, L., Tennenbaum, T., and Yuspa, S. H. (1996) J. Invest. Dermatol. 106, 254–260.
Thin-Layer Affinity Chromatography in Analysis of Protein–Ligand Affinity Mika P. A. Laitinen,1 Kirsi M. Sojakka, and Matti Vuento Department of Biological and Environmental Science, University of Jyva¨skyla¨, Vapaudenkatu 4, FIN-40100 Jyva¨skyla¨, Finland Received June 19, 1996
Work with proteins often involves operations that can induce conformational changes and affect the bind1
To whom correspondence should be addressed. Fax: /358 (0)14 602 221. ANALYTICAL BIOCHEMISTRY ARTICLE NO.
ing of protein to its ligand. Hence, it is important to be able to monitor the affinity state of the protein studied relative to the original native state. Quantitative methods for assessing affinity parameters involve setting up equilibrium mixtures of protein and ligand and analyzing the concentrations of bound and free species by various physicochemical methods (1). From such measurements, the dissociation constant of the binding reaction can usually be derived. Unfortunately, these methods are tedious and do not allow a rapid check to be made on the affinity state of the protein. Affinity chromatography has been previously used for the estimation of dissociation constants (2, 3). Recent developments in affinity chromatography have introduced thin-layer techniques in the rapid analysis of antigen binding to antibody (4–7), analogous to protein–ligand interaction. In particular, Lou et al. (5) used several discrete antibody zones immobilized on membrane to analyze lipoprotein concentration in serum. That assay was based on a competition of lipoprotein present in a sample with lipoprotein labeled with colloidal selenium for binding to the antibody zones. These developments led us to test the use of thin-layer affinity chromatography in studying the affinity of protein–ligand interaction after perturbation of protein by denaturation. The method was used in determination of the transition temperature of heat-denatured fibronectin and heatdenatured succinylated avidin on the basis of their binding to polyclonal antibody and to biotin, respectively. On a strip of nitrocellulose membrane, a capture region comprising eight discrete capture zones was set up by allowing adsorption of the ligand or protein-conjugated ligand onto the membrane. The protein in its reference affinity state was labeled with colloidal gold, and the labeled conjugate was allowed to comigrate with nonlabeled protein through the capture region. During migration, the labeled and nonlabeled protein competed for binding to the ligand. The degree to which the discrete capture zones bound the labeled reference protein was dependent on the affinity state of the studied protein. Materials and Methods Nitrocellulose membrane of a nominal pore size of 12 mm was obtained from Sleicher and Schuell (Darmstadt, Germany). Succinic anhydride, ovalbumin, Sacetylmercaptosuccinic anhydride, N-succinimidyl-4( N - maleimidomethyl ) - cyclohexane - 1 - carboxylate (SMCC),2 and biotinamidocaproate N-hydroxysuccinimide ester were obtained from Sigma Chemical Co. (St. 2 Abbreviations used: SMCC, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate; HABA, 4*-hydroxyazobenzene-2carboxylic acid.
243, 279–282 (1996)
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0003-2697/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
11-23-96 01:59:39
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