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A filter paper sandwich method using small volumes of reagents for the detection of antigens electrophoretically transferred onto nitrocellulose
Journal oflmmunologicalMethods, 75 (1984) 333-338 Elsevier
333
JIM03319
A Filter Paper Sandwich Method Using Small Volumes of Reagents for the Detection of Antigens Electrophoretically Transferred onto Nitrocellulose Gordon C. Douglas 1 and Barry F. King Department of Human Anatomy, School of Medicine, University of California, Davis, CA 95616, U.S.A. (Received 22 August 1984, accepted 10 September 1984)
A method is described which allows antigens electrophoretically 'blotted' onto nitrocellulose to be probed using small volumes ( < 0.3 ml) of antibody solutions. Reagent volumes required for this method are more than 10-fold lower than the minimum volumes attainable using either custom made or commercial incubation trays. The procedure is simple and economical and should be applicable to any situation where it is desirable to either conserve or reduce the amount of screening reagent used.
Key words: antigenic analysis - immunoblotting - transferrin receptor
Introduction
The electrophoretic transfer of proteins from SDS-polyacrylamide gels onto nitrocellulose (Western blotting) (Towbin et al., 1979) is seeing widespread application in many laboratories. After transfer, specific proteins are located by incubating the nitrocellulose sheets or strips with appropriate polyclonal or monoclonal antibodies followed by incubation with a second antibody or protein A to which has been conjugated either ~25I, horseradish peroxidase or alkaline phosphatase (Batteiger et al., 1982; Ogata et al., 1983; Blake et al., 1984). Exposure of the nitrocellulose to these probes is carried out in dishes or trays and, depending on the number and size of strips being tested, can expend relatively large volumes (20-60 ml) of reagents. While this is not a problem if laboratory-produced antibodies or inexpensive commercial preparations are easily obtainable, wastage can become restrictive when antibody availability is limited. Under the latter circumstances, and where radioactive probes are used, it is clearly advantageous to keep incubation volumes to a minimum. Tsang et al. (1983) have shown that volumes of about 5 ml can be used successfully if narrow strips of nitrocellulose and correspondingly narrow incubation I To whom correspondence should be addressed. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
334
trays are utilized. Such an incubation tray has recently become available commercially. Recently, during the course of our studies, it became necessary to identify the transferrin receptor following electrophoretic blotting of placental microvillous membrane proteins. While successful results were obtained using a commercial monoclonal anti-transferrin receptor IgG as first antibody, it quickly became apparent that the high cost of this material would restrict its routine use, even when incubation volumes were reduced to 5 ml. Accordingly, we developed a method in which volumes of less than 0.3 ml can be used to probe a nitrocellulose strip. The method is described here.
Materials and Methods
General materials Vertical slab gel electrophoresis equipment was purchased from Hoeffer Scientific Instruments, San Francisco, CA and electroblotting equipment was from Bio-Rad Laboratories, Richmond, CA. Bio-Rad Laboratories also supplied the electrophoresis reagents, affinity-purified goat anti-mouse IgG (peroxidase-conjugated), peroxidase color development reagent, molecular weight standards and the nitrocellulose membrane. Mouse monoclonal anti-human transferrin receptor was obtained from Boehringer Mannheim Biochemicals, IN. Tween-20 was obtained from Sigma Chemical Co., St. Louis, MO.
SDS-polyacrylamide gel electrophoresis and transfer onto nitrocellulose A microvillous plasma membrane fraction, prepared from term human placenta as described by Smith et al. (1977), was used as the antigen source in the present study. Prior to electrophoresis, microvillous membrane was solubilized in sample buffer (40 nM Tris-HC1, pH 6.8, 2% (w/v) SDS, 0.8% ( w / v ) DTT, 5% (v/v) glycerol and 0.002% (w/v) bromophenol blue) and heated at 100°C for 5 min. Vertical slab SDS-polyacrylamide gel electrophoresis was carried out using the discontinuous buffer system described by Laemmli (1970). A 3% stacking gel and 7.5% separating gel were used. The dimensions of the separating gel were 11 cm x 14 cm. Samples were electrophoresed at 75 V until the tracking dye reached the separating gel, and then at 150 V until the dye was within 1 cm of the bottom of the gel. Proteins were immediately transferred from the gel onto a sheet of nitrocellulose (Towbin et al., 1979). A Bio-Rad Trans-Blot apparatus was used and transfer was at 0.25 A for 3 h. The buffer system consisted of 25 mM Tris-HC1, pH 8.3, 192 mM glycine, 20% (v/v) methanol. Following transfer, the nitrocellulose sheet was marked with a pencil to allow subsequent alignment and orientation and then cut into 0.5 cm wide strips. The strips were either used immediately for analysis or were blotted dry on filter paper and stored in airtight boxes at - 2 0 ° C until needed. (We have found that strips can be stored in this way for at least 6 months with no apparent detrimental effects on bound antigens.)
335
Localization of nitrocellulose-bound transferrin receptor Strips were first of all blocked by incubation in 0.1 M Tris-HC1, pH 7.3, containing 0.15 NaC1 and 0.05% Tween-20 for 1 h at 37°C, as recommended by Batteiger et al. (1982). Strips were then incubated with the first antibody - - in this case a mouse monoclonal anti-human transferrin receptor antibody. In order to facilitate incubation with small volumes of antibody the following method was devised. Two identical strips of Whatman no. 1 filter paper were prepared of a size slightly larger than the nitrocellulose strip. The filter papers were held with forceps and allowed to absorb the monoclonal antibody (diluted to 80 t~g/ml in Tris-saline-Tween buffer) which was slowly applied with the aid of an automatic pipette. A total volume of 0.25 ml is sufficient to saturate 2 filter paper strips measuring 11.5 c m x 0.75 cm. The nitrocellulose strip was then 'sandwiched' between the 2 pieces of pre-soaked filter paper which were then placed between two glass plates (10 cm x 10 cm) and held tightly together using spring clips (Fig. 1). The glass plate assembly was then placed in an airtight, humid box and incubated at 37°C for 1 h and at 4°C overnight. The nitrocellulose strip was then removed from the filter paper sandwich, washed twice, each time for 10 min, in Tris-saline-Tween buffer and then incubated with affinity-purified HRP-conjugated goat anti-mouse IgG (diluted 1 : 2 0 0 0 in Tris-saline-Tween Buffer) for 2 h at 37°C. This incubation was carried out in plastic dishes containing 50 ml antibody solution and with gentle agitation. After 2 final washes, each for 10 min in Tris-saline-Tween buffer, the strip was rinsed briefly with deionized water and incubated in an HRP-color development solution until reaction product became visible (2-10 min) and then finally rinsed in distilled water. The strips were photographed while still wet.
Fig. 1. This shows the final glass plate assemblycontaining the filter paper/nitrocellulose 'sandwich'.
336 Some strips were stained for p r o t e i n in a solution of 0.1% ( w / v ) a m i d o black 10-B in 45% ( v / v ) m e t h a n o l / 1 0 % ( v / v ) acetic acid.
Results and Discussion In o r d e r to illustrate the use of the m e t h o d d e s c r i b e d in this paper, we a t t e m p t e d to identify the transferrin r e c e p t o r in a h u m a n p l a c e n t a l microvillous m e m b r a n e p r e p a r a t i o n after e l e c t r o b l o t t i n g o n t o nitrocellulose. The h u m a n transferrin r e c e p t o r is a d i m e r c o m p o s e d of 2 identical subunits, each with a m o l e c u l a r mass of a b o u t 90,000 D a (Bleil a n d Bretscher, 1982). Fig. 2C shows a nitrocellulose strip which had been i n c u b a t e d with m o n o c l o n a l a n t i - t r a n s f e r r i n r e c e p t o r a n t i b o d y followed by p e r o x i d a s e - c o n j u g a t e d goat a n t i - m o u s e IgG. The transferrin r e c e p t o r s u b u n i t is readily identified as the intense b a n d of reaction p r o d u c t c o r r e s p o n d i n g to a species
A
B
C
D
10-3x Mr 92.5
i
66.2
I l
45
31
21.5
•
m
,,u
TD Fig. 2. Immunochemical staining and amido black staining of microvillous membrane proteins electroblotted onto nitrocellulose. A: amido black 10 B staining of protein standards. B. Amido black 10-B staining of microvillous membrane proteins. C: strip incubated with monoclonal mouse-anti-transferrin receptor antibody followed by peroxidase-conjugated goat anti-mouse lgG and then stained for peroxidase activity as described in the methods section. D: details as for C except that incubation with the mouse anti-transferrin receptor was omitted. The position of the tracking dye is indicated by TD.
337 with an apparent molecular mass of approximately 90,000 Da. This species can also be seen on a strip stained for protein (Fig. 2B) where it appears as a relatively minor component of the microvillous membrane preparation. A faint band of reaction product can be seen near the top of the strip in Fig. 2C: this probably represents undissociated receptor. The method described here allows the immunological identification of antigens bound to nitrocellulose with minimal wastage of precious reagents. Using a nitrocellulose strip 0.5 cm X 10 cm and appropriately sized pieces of filter paper, only 250 ~1 of antibody solution is required for incubation. Where reagents are not limiting, incubations are usually carried out in dishes using volumes between 20-60 ml. Using long narrow troughs, as suggested by Tsang et al. (1983), volumes can be reduced to about 5 ml. The procedure outlined in this paper therefore offers a considerable advantage over these other methods in situations where the conservation of a particular reagent is desirable. Volumes could be reduced even more by using smaller nitrocellulose/filter paper strips. Depending on the size of the glass plates used, several strips can be incubated simultaneously, with the precaution that, if different antigens are being screened, the strips are not allowed to touch one another. Although for our purposes it was only necessary to use the filter paper overlay method for incubation with the first antibody, the same technique can be used for the second antibody incubation if required. It may be particularly useful, for example, in those situations in which 125I-labeled protein A is used as the second 'antibody'. As well as conserving reagents, the method eliminates the risk of spillage and would considerably minimize exposure to radioactivity. Although we find it convenient to expose strips to antibody-soaked filter paper for 2 h at 37°C and then at 4°C overnight, perfectly satisfactory results were obtained by omitting the overnight incubation at 4°C. With low amounts of antigen, however, the additional overnight incubation may increase the sensitivity. When compared under the same conditions of time and temperature the filter paper method yielded results which were visually indistinguishable from those obtained using the conventional immersion method, indicating no major loss of sensitivity. Finally, the method described uses inexpensive materials found in most laboratories and therefore provides a considerable saving over expensive, small volume (5 ml) incubation trays which have recently become available.
Acknowledgement This work was supported by Grant H D 11658 from the National Institutes of Health.
References Batteiger, B., W.J. Newhall and R.B. Jones, 1982, J. Immunol. Methods 55, 297. Blake, M.S., K.H, Johnston, G.J. Russell-Jones and E.C. Gotschlich, 1984, Anal. Biochem. 136, 175.
338 Bleil, J,D. and M.S. Bretscher, 1982, EMBO J. 1,351. Laemmli, U.K., 1970, Nature (London) 227, 680, Ogata, K., M. Arakawa, T. Kasahara, K. Shioiri-Nakano and K. Hiraoka, 1983, J. Immunol. Methods 65, 75. Smith, C.H., D.M. Nelson, B.F. King, T.M. Donohue, S.M. Ruzycki and L.K. Kclley, 1977, Am. J. Obstet. Gynecol. 128, 190. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Tsang, V., J. Peralta and A.R. Simons, 1983, Methods Enzymol. 92, 377.
333
JIM03319
A Filter Paper Sandwich Method Using Small Volumes of Reagents for the Detection of Antigens Electrophoretically Transferred onto Nitrocellulose Gordon C. Douglas 1 and Barry F. King Department of Human Anatomy, School of Medicine, University of California, Davis, CA 95616, U.S.A. (Received 22 August 1984, accepted 10 September 1984)
A method is described which allows antigens electrophoretically 'blotted' onto nitrocellulose to be probed using small volumes ( < 0.3 ml) of antibody solutions. Reagent volumes required for this method are more than 10-fold lower than the minimum volumes attainable using either custom made or commercial incubation trays. The procedure is simple and economical and should be applicable to any situation where it is desirable to either conserve or reduce the amount of screening reagent used.
Key words: antigenic analysis - immunoblotting - transferrin receptor
Introduction
The electrophoretic transfer of proteins from SDS-polyacrylamide gels onto nitrocellulose (Western blotting) (Towbin et al., 1979) is seeing widespread application in many laboratories. After transfer, specific proteins are located by incubating the nitrocellulose sheets or strips with appropriate polyclonal or monoclonal antibodies followed by incubation with a second antibody or protein A to which has been conjugated either ~25I, horseradish peroxidase or alkaline phosphatase (Batteiger et al., 1982; Ogata et al., 1983; Blake et al., 1984). Exposure of the nitrocellulose to these probes is carried out in dishes or trays and, depending on the number and size of strips being tested, can expend relatively large volumes (20-60 ml) of reagents. While this is not a problem if laboratory-produced antibodies or inexpensive commercial preparations are easily obtainable, wastage can become restrictive when antibody availability is limited. Under the latter circumstances, and where radioactive probes are used, it is clearly advantageous to keep incubation volumes to a minimum. Tsang et al. (1983) have shown that volumes of about 5 ml can be used successfully if narrow strips of nitrocellulose and correspondingly narrow incubation I To whom correspondence should be addressed. 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
334
trays are utilized. Such an incubation tray has recently become available commercially. Recently, during the course of our studies, it became necessary to identify the transferrin receptor following electrophoretic blotting of placental microvillous membrane proteins. While successful results were obtained using a commercial monoclonal anti-transferrin receptor IgG as first antibody, it quickly became apparent that the high cost of this material would restrict its routine use, even when incubation volumes were reduced to 5 ml. Accordingly, we developed a method in which volumes of less than 0.3 ml can be used to probe a nitrocellulose strip. The method is described here.
Materials and Methods
General materials Vertical slab gel electrophoresis equipment was purchased from Hoeffer Scientific Instruments, San Francisco, CA and electroblotting equipment was from Bio-Rad Laboratories, Richmond, CA. Bio-Rad Laboratories also supplied the electrophoresis reagents, affinity-purified goat anti-mouse IgG (peroxidase-conjugated), peroxidase color development reagent, molecular weight standards and the nitrocellulose membrane. Mouse monoclonal anti-human transferrin receptor was obtained from Boehringer Mannheim Biochemicals, IN. Tween-20 was obtained from Sigma Chemical Co., St. Louis, MO.
SDS-polyacrylamide gel electrophoresis and transfer onto nitrocellulose A microvillous plasma membrane fraction, prepared from term human placenta as described by Smith et al. (1977), was used as the antigen source in the present study. Prior to electrophoresis, microvillous membrane was solubilized in sample buffer (40 nM Tris-HC1, pH 6.8, 2% (w/v) SDS, 0.8% ( w / v ) DTT, 5% (v/v) glycerol and 0.002% (w/v) bromophenol blue) and heated at 100°C for 5 min. Vertical slab SDS-polyacrylamide gel electrophoresis was carried out using the discontinuous buffer system described by Laemmli (1970). A 3% stacking gel and 7.5% separating gel were used. The dimensions of the separating gel were 11 cm x 14 cm. Samples were electrophoresed at 75 V until the tracking dye reached the separating gel, and then at 150 V until the dye was within 1 cm of the bottom of the gel. Proteins were immediately transferred from the gel onto a sheet of nitrocellulose (Towbin et al., 1979). A Bio-Rad Trans-Blot apparatus was used and transfer was at 0.25 A for 3 h. The buffer system consisted of 25 mM Tris-HC1, pH 8.3, 192 mM glycine, 20% (v/v) methanol. Following transfer, the nitrocellulose sheet was marked with a pencil to allow subsequent alignment and orientation and then cut into 0.5 cm wide strips. The strips were either used immediately for analysis or were blotted dry on filter paper and stored in airtight boxes at - 2 0 ° C until needed. (We have found that strips can be stored in this way for at least 6 months with no apparent detrimental effects on bound antigens.)
335
Localization of nitrocellulose-bound transferrin receptor Strips were first of all blocked by incubation in 0.1 M Tris-HC1, pH 7.3, containing 0.15 NaC1 and 0.05% Tween-20 for 1 h at 37°C, as recommended by Batteiger et al. (1982). Strips were then incubated with the first antibody - - in this case a mouse monoclonal anti-human transferrin receptor antibody. In order to facilitate incubation with small volumes of antibody the following method was devised. Two identical strips of Whatman no. 1 filter paper were prepared of a size slightly larger than the nitrocellulose strip. The filter papers were held with forceps and allowed to absorb the monoclonal antibody (diluted to 80 t~g/ml in Tris-saline-Tween buffer) which was slowly applied with the aid of an automatic pipette. A total volume of 0.25 ml is sufficient to saturate 2 filter paper strips measuring 11.5 c m x 0.75 cm. The nitrocellulose strip was then 'sandwiched' between the 2 pieces of pre-soaked filter paper which were then placed between two glass plates (10 cm x 10 cm) and held tightly together using spring clips (Fig. 1). The glass plate assembly was then placed in an airtight, humid box and incubated at 37°C for 1 h and at 4°C overnight. The nitrocellulose strip was then removed from the filter paper sandwich, washed twice, each time for 10 min, in Tris-saline-Tween buffer and then incubated with affinity-purified HRP-conjugated goat anti-mouse IgG (diluted 1 : 2 0 0 0 in Tris-saline-Tween Buffer) for 2 h at 37°C. This incubation was carried out in plastic dishes containing 50 ml antibody solution and with gentle agitation. After 2 final washes, each for 10 min in Tris-saline-Tween buffer, the strip was rinsed briefly with deionized water and incubated in an HRP-color development solution until reaction product became visible (2-10 min) and then finally rinsed in distilled water. The strips were photographed while still wet.
Fig. 1. This shows the final glass plate assemblycontaining the filter paper/nitrocellulose 'sandwich'.
336 Some strips were stained for p r o t e i n in a solution of 0.1% ( w / v ) a m i d o black 10-B in 45% ( v / v ) m e t h a n o l / 1 0 % ( v / v ) acetic acid.
Results and Discussion In o r d e r to illustrate the use of the m e t h o d d e s c r i b e d in this paper, we a t t e m p t e d to identify the transferrin r e c e p t o r in a h u m a n p l a c e n t a l microvillous m e m b r a n e p r e p a r a t i o n after e l e c t r o b l o t t i n g o n t o nitrocellulose. The h u m a n transferrin r e c e p t o r is a d i m e r c o m p o s e d of 2 identical subunits, each with a m o l e c u l a r mass of a b o u t 90,000 D a (Bleil a n d Bretscher, 1982). Fig. 2C shows a nitrocellulose strip which had been i n c u b a t e d with m o n o c l o n a l a n t i - t r a n s f e r r i n r e c e p t o r a n t i b o d y followed by p e r o x i d a s e - c o n j u g a t e d goat a n t i - m o u s e IgG. The transferrin r e c e p t o r s u b u n i t is readily identified as the intense b a n d of reaction p r o d u c t c o r r e s p o n d i n g to a species
A
B
C
D
10-3x Mr 92.5
i
66.2
I l
45
31
21.5
•
m
,,u
TD Fig. 2. Immunochemical staining and amido black staining of microvillous membrane proteins electroblotted onto nitrocellulose. A: amido black 10 B staining of protein standards. B. Amido black 10-B staining of microvillous membrane proteins. C: strip incubated with monoclonal mouse-anti-transferrin receptor antibody followed by peroxidase-conjugated goat anti-mouse lgG and then stained for peroxidase activity as described in the methods section. D: details as for C except that incubation with the mouse anti-transferrin receptor was omitted. The position of the tracking dye is indicated by TD.
337 with an apparent molecular mass of approximately 90,000 Da. This species can also be seen on a strip stained for protein (Fig. 2B) where it appears as a relatively minor component of the microvillous membrane preparation. A faint band of reaction product can be seen near the top of the strip in Fig. 2C: this probably represents undissociated receptor. The method described here allows the immunological identification of antigens bound to nitrocellulose with minimal wastage of precious reagents. Using a nitrocellulose strip 0.5 cm X 10 cm and appropriately sized pieces of filter paper, only 250 ~1 of antibody solution is required for incubation. Where reagents are not limiting, incubations are usually carried out in dishes using volumes between 20-60 ml. Using long narrow troughs, as suggested by Tsang et al. (1983), volumes can be reduced to about 5 ml. The procedure outlined in this paper therefore offers a considerable advantage over these other methods in situations where the conservation of a particular reagent is desirable. Volumes could be reduced even more by using smaller nitrocellulose/filter paper strips. Depending on the size of the glass plates used, several strips can be incubated simultaneously, with the precaution that, if different antigens are being screened, the strips are not allowed to touch one another. Although for our purposes it was only necessary to use the filter paper overlay method for incubation with the first antibody, the same technique can be used for the second antibody incubation if required. It may be particularly useful, for example, in those situations in which 125I-labeled protein A is used as the second 'antibody'. As well as conserving reagents, the method eliminates the risk of spillage and would considerably minimize exposure to radioactivity. Although we find it convenient to expose strips to antibody-soaked filter paper for 2 h at 37°C and then at 4°C overnight, perfectly satisfactory results were obtained by omitting the overnight incubation at 4°C. With low amounts of antigen, however, the additional overnight incubation may increase the sensitivity. When compared under the same conditions of time and temperature the filter paper method yielded results which were visually indistinguishable from those obtained using the conventional immersion method, indicating no major loss of sensitivity. Finally, the method described uses inexpensive materials found in most laboratories and therefore provides a considerable saving over expensive, small volume (5 ml) incubation trays which have recently become available.
Acknowledgement This work was supported by Grant H D 11658 from the National Institutes of Health.
References Batteiger, B., W.J. Newhall and R.B. Jones, 1982, J. Immunol. Methods 55, 297. Blake, M.S., K.H, Johnston, G.J. Russell-Jones and E.C. Gotschlich, 1984, Anal. Biochem. 136, 175.
338 Bleil, J,D. and M.S. Bretscher, 1982, EMBO J. 1,351. Laemmli, U.K., 1970, Nature (London) 227, 680, Ogata, K., M. Arakawa, T. Kasahara, K. Shioiri-Nakano and K. Hiraoka, 1983, J. Immunol. Methods 65, 75. Smith, C.H., D.M. Nelson, B.F. King, T.M. Donohue, S.M. Ruzycki and L.K. Kclley, 1977, Am. J. Obstet. Gynecol. 128, 190. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Tsang, V., J. Peralta and A.R. Simons, 1983, Methods Enzymol. 92, 377.