Journal of Biochemical and Biophysical Methods, 21 (1990) 129-144
129
Elsevier BBM 00824
Quantitation of immobilized proteins * William S. Lewis 1 and Sheldon M. Schuster 2 1 Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE and 2 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, U.S.A.
(Received 8 February 1990) (Accepted 5 March 1990)
Summary A method for the quantitative determination of immobilized proteins based on the binding and subsequent elution of Coomassie Blue R is presented. Also presented is a method for the immobilization of proteins in solution by entrapment in polyacrylamide. These entrapped proteins are then available for use in the assay method presented. Other analytical procedures can also be performed on the entrapped proteins, either alone or in combination with the protein quantitation. The dye binding and elution method presented provides a sensitive and, in most applications, rapid method for the quantitative detection of immobilized proteins. Rather than immobilization being an obstacle to the assay method, this approach utilizes the advantages of immobilization for the removal of excess reagents. Application of this approach to several types of immobilized protein are presented.
Key words: Protein; Quantitation
Introduction Immobilization of proteins on or within solid-phase supports provides a convenient method for the manipulation of proteins and facilitates the removal of soluble
* This work was supported in part by a grant from Finnsugar Biochemicals, Inc.
Abbreviations: TEMED, N,N,N',N'-tetramethylethylenediamine; TCA, trichloroacetic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; Coomassie Blue, Coomassie Brilliant Blue R-250; Bis, N, N '-methylenebisacrylamide.
Correspondence address: Sheldon M. Schuster, Interdisciplinary Center for Biotechnology Research, 1301 Fifield Hail, University of Florida, Gainesville, FL 32611, U.S.A. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
130 components from the immobilized protein. For most applications, the amount of protein attached to the support must be determined. Several methods have been developed for this purpose [1-3]. Since determinations are expressed as the amount of protein bound per unit volume of the support material, measurement of both the amount of protein in a sample and the volume of the support in the sample must be determined. Typically, the support is packed into a column and the volume of the packed column is measured. Then the amount of protein bound is determined either by the difference between the amount added and the amount remaining in the solution, following coupling or by one of several detection methods [1,2]. An alternative method is to pack the support into a cuvette and to measure the absorbance of the protein in the packed support [3]. These methods are tedious and require that relatively large volumes of the immobilized protein be available. In addition, little standardization of the pressure under which the support is packed is used, leading to large variations in the support volume determinatiofi. Here we report a method for the determination of the concentration of immobilized proteins. This method utilizes the binding and subsequent elution of a dye similar to several other systems which have previously been reported for proteins in solution [4-8]. The protein in the present method is immobilized during the staining, facilitating the removal of excess dye from the protein prior to elution of the protein-bound dye. Application of this method to proteins immobilized by several methods is reported. A method for the immobilization of proteins by addition of acrylamide to the solution and polymerization of the acrylamide is also presented. This method is applied, in conjunction with the method for the quantitation of immobilized proteins, for the removal of substances in the solution which interfere with the protein quantitation. A number of methods have been developed for the determination of total protein concentration in solutions [4-12]. All of these methods, however, exhibit some interference by reagents commonly found in the sample solutions [13-14]. Modifications of these procedures have resulted in numerous adaptations which eliminate one or a small group of these interferences [6,15]. A general method has yet to be developed for the assay of protein concentrations in samples free of interference by all of these substances. Since many samples for protein assays contain varying combinations of these substances, methods for the elimination of any one interfering substance are not generally applicable. Techniques for the removal of interfering reagents, such as dialysis, are inconvenient because of the small volumes and large number of samples involved and lack of quantitative recovery of the sample. Repeated precipitation of the protein with trichloroacteic acid is often used to remove interfering substances. This method is limited by low recovery of the protein and difficulty in dissolving the precipitated protein. This method can be utilized as an alternative to dialysis or other methods of reagent exchange. The proteins treated in this way can later be used for other analytical procedures, methods for many of which have been developed for acrylamide embedded proteins resulting from gel electrophoresis [16-18] and can be applied with little or no modification.
131 Materials and Methods Materials Methanol was obtained from Fisher (Fairlawn, N J) and distilled in glass. Coomassie Blue (C.I. 42660), Amido Black (C.I. 20470), Fast Green F C F (C.I. 42053), acrylamide, TEMED, .Bis and ammonium persulfate were Electrophoresis Grade obtained from Bio-Rad (Richmond, CA). Tissue culture plates (24 well) were from Sargent-Welch (Skokie, IL). HPLC grade pyridine was from Aldrich (Milwaukee, WI). Ammonium sulfate was from EM Science (Cherry Hill, N J). Sucrose was obtained from Schwarz/Mann Biotechnologies (Cleveland, OH), and chymotrypsin was from Worthington Biochemicals (Freehold, N J). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO). Immobilization methods BSA was coupled to Sepharose 4B-CL by the CNBr method of Cuatrecasas [19]. The resin was activated in a single batch. The activated resin was then divided into approximately equal aliquots (10 ml packed resin each). Different amounts of BSA were added to the resin in a total volume of 10 ml of 0.1 M sodium phosphate buffer, p H 9.5. Following coupling at 4 ° C for 24 h, the remaining reactive sites on the resin were blocked by addition of ethanolamine to 1 M. The resin was removed by filtration and washed with 2-3 vol. of buffer. The filtrate and wash solutions were combined and the unbound protein was determined by measurement of the volume and absorbance of the combined solutions. Bound protein was calculated by the difference in absorbance between the protein solution added to the resin and the unbound protein. The volume of the substituted resin was determined by packing the resin into a graduated column. The concentration of the protein on the resin was determined by the amount of protein bound to the resin and the resulting volume of the resin. Immobilization of BSA on cellulose nitrate filters was accomplished by spotting Jifferent amounts of a stock BSA solution (1 m g / m l ) into each well of a Bio-Rad Dot-Blot apparatus. When all of the liquid was absorbed into the filter, a vacuum was applied and each well was washed with two 100 /~1 aliquots of 0.1 M sodium phosphate buffer, p H 6.8. Protein standards and standard protein assays Protein standards were made by carefully weighing out the desired amounts of protein and dissolving them in water. A single stock solution of each protein was used for all of the assay methods tested. Typically, stock solutions were used at a concentration of 1 mg/ml. Protein concentrations were determined by the methods of Lowry et al. [9] and Bradford [10] where indicated in the text. The method used for the Bradford determination was according to the standard assay protocol provided in the Bio-Rad Protein assay *. Absorbances of dye solutions were
* Bio-Rad protein assay, bulletin 1069 Bio-Rad Laboratories, Richmond, CA, U.S:A.
132 measured in disposable plastic semimicro cuvettes (4 x 10 m m with a 10 m m light path) obtained from Sarstadt using a Beckman DU-50 spectrophotometer. Radioactive resins were made by coupling of [14C]serine to a glycyl-Sepharose resin. Preparation of the resin was as described by Cuatrecasas for the coupling of alanine [19]. Coupling of the serine to the resin was accomplished by activation of the resin with D C C D to form the activated N-hydroxysuccinimide ester as described. For embedding of proteins in solution by entrapment in acrylamide, the following method was used. Protein samples containing the indicated amount of protein, and any other reagents as indicated, were diluted with water to a total volume of 50 #1 and placed in a 75 x 10 m m glass test tube. To each sample was added 25 #1 of a solution of 30% acrylamide * *, 0.8% Bis. Then 1.5 #1 of a freshly prepared solution of 10% a m m o n i u m persulfate was added to each sample. The samples were then mixed thoroughly and 1 #1 of T E M E D was added to each. The samples were left at room temperature for 10 min to allow complete polymerization. Gel electrophoresis of samples containing 0 - 1 0 #g of BSA were done using the Tris-borate gel system described by Sykes and Bailey [20]. Electrophoresis at 150 V was continued until the bromophenol Blue tracking dye reached the bottom of the gel slab.
Assay of immobilized proteins Sepharose immobilized proteins.
A portion of the resin suspension to be assayed is placed in a microcentrifuge tube. The resin is pelleted by centrifugation at 3000 r p m for 2 min. The resin is suspended in 1 ml of the staining solution which consists of 0.25% Coomassie Blue R, 30% methanol, and 10% acetic acid. After 10 min at room temperature, the sample is centrifuged for 3 min at 3000 rpm. The resin is suspended in 1 ml of the staining solution and incubated at room temperature for another 10 min. The sample is then centrifuged at 3000 rpm for 3 min and the supernatant fluid is removed. A small portion of the stained resin pellet, about 5 - 1 0 /xl, is carefully scooped out with a spatula and placed in another microfuge tube containing 1 ml of the destaining solution composed of 30% methanol and 7.5% acetic acid. After a few seconds of mixing to insure that the pellet is well suspended in the solution, the sample is centrifuged for 3 min at 5000 rpm. The supernatant fluid is decanted and saved for determination of the resin volume. The bound dye is eluted by suspending the pellet in 0.4 ml of a solution containing 1% SDS and 1% sodium bicarbonate. To this suspension is added 0.6 ml of methanol and the solution is mixed well to ensure thorough suspension of the resin. The resin is pelleted by centrifugation at 5000 r p m for 3 min and the supernatant fluid is decanted and saved for determination of the bound protein content of the resin sample. The amounts of dye in the supernatant fluids from the elution and destaining steps is determined by measurement of their absorbance at 595 nm. In the event
* * Percent solutions are expressed as weight/volume when the solute is a solid and volume/volume when the solute is a liquid at room temperature.
133 that the resin pellets become unstable during any of the decanting steps above, the preceding centrifugation step can be repeated to repack the pellet for complete removal of the supernatant fluid. Cellulose nitrate filter immobilized proteins. The filter containing the immobilized proteins is stained by incubation in a solution of 0.125% Coomassie Blue R, 30% methanol and 7.5% acetic.acid for 15 min with good mixing. The stained filter is then destained by several incubations with 30% methanol, 7.5% acetic acid until the solution remains colorless. The regions containing the protein to be assayed are then cut out with scissors and placed in separate wells of a 24-well tissue culture plate. The bound dye is eluted by incubation with 0.4 ml 1% SDS, 1% sodium bicarbonate, with good mixing for 30 min. The incubation is continued for an additional 60 min following addition of 0.6 ml methanol to each well. The amount of dye released is determined by measurement of the absorbance of the resulting solution at 595 nm. A crylamide embedded proteins. The gel containing the protein sample was removed from the test tube with a spatula and each sample was placed in a separate well of a 24-well tissue culture plate. The samples were incubated with the following sequence of reagents for at least 2 h each step. During incubation, mixing was accomplished by placing the plate on a benchtop shaker (Tektator V, S / P products, Evanston, IL). The wash solutions used were: - two washes with 10% trichloroacetic acid three washes with 10% acetic acid three washes with a solution of 5% methanol, 7.5% acetic acid two washes with a solution of 30% methanol, 7.5% acetic acid The samples were stained with 0.125% Coomassie Blue R in 35% methanol, 7.5% acetic acid for at least 6 h. The samples were destained with 30% methanol, 7.5% acetic acid using several changes in 2 ml portions. The samples were destained until blanks (samples containing no protein) appeared colorless. Typically the destaining required three changes of solution. When the samples were destained, the dye bound to the protein was eluted. In order to prevent evaporation during the elution step, a gasket was cut from a sheet of rubber to fit inside the lid of the tissue culture plate. The lid, with the gasket, was held in place by two rubber bands. As an additional precaution, the plate was sealed in a plastic reclosable sandwich bag. Except where noted, the following procedure was used for elution of the bound dye. The samples were incubated in 0.6 ml of a solution containing 1% SDS, 1% sodium bicarbonate for at least 2 h. Then 0.9 ml of methanol was added and incubation was continued for several more hours. This elution method was adapted from the protein assay described by Nakao et al. [6]. The combined elution solution (1.5 ml total volume) was collected and the absorbance of the eluted dye determined. Alternative methods for polyacrylamide embedded proteins. As described in Resuits, some samples were stained with Amido Black. For these samples, 1% Amido Black in 30% methanol, 7.5% acetic acid was used instead of the Coomassie Blue solution described above. All other steps remained the same. Alternative elution methods were also investigated. Two different methods were -
-
-
134 tested, elution with methanol and elution with pyridine. In samples eluted with methanol, the samples were eluted by incubating with 0.5 ml of 45% methanol, 10% acetic acid for 1 h. Then 0.5 ml of 80% methanol, 7.5% acetic acid was added to the well containing the gel and the first ehition solution. Following incubation for an additional hour, 0.5 ml of methanol was added and the incubation was continued for several more hours. This stepwise increase in the concentration of methanol was found to be necessary to insure complete elution of the dye from the interior of the gel. The resulting elution solution (1.5 ml total volume) was collected and the absorbance of the eluted dye was determined. Some samples were eluted from the gel pieces by incubation for several hours in 1.5 ml of a 25% pyridine solution. This method was adapted from Fenner et al. [21]. The elution solution was then removed and the absorbance of the eluted dye determined. Gel electrophoresis. Sections of the gel containing the protein bands or sections of varying size containing no protein were cut from the gel with a razor blade. Excess solution was removed by blotting the section dry with tissue paper and the section was weighed. The dye was then eluted from the gel as described previously except that the volumes were adjusted proportionally so that the total volume of the elution solution was 1.0 ml.
Results
Sepharose-immobilized proteins Application of the assay method to Sepharose-immobilized proteins involves the determination of both the volume of the resin in the sample a n d the amount of protein bound to the resin. The determination of the volume of resin used in the assay assumes that the volume of the bulk solution in and between the resin beads is proportional to the volume of resin present. The volume is then determined by the amount of dye removed from the resin in the destaining step. In order to test this assumption, a resin was synthesized to contain an immobilized 14C-labeled amino acid. A portion of this resin was packed in a 0.2 ml pipette which had been cut to produce a graduated column. The end of the pipette was covered by a piece of nylon mesh to allow the liquid portion of the resin suspension to pass through while retaining the resin. The volume of the packed resin was determined by the graduations of the pipette. This packed resin was then monitored to determine the radioactivity per ml of packed resin. Other portions of the resin were stained by the presented assay method and the amount of stain released in the destaining step was determined by its absorbance. The volume of resin in each sample was determined from the radioactivity present in the sample. The results of this experiment are shown in Fig: 1. The volume of dye released in the staining step is proportional to the volume of resin present. The amount of dye released in the elution step provides a measure of the amount of protein bound to the resin used in the sample. The amount of protein bound per unit volume of resin is proportional to the ratio of absorbances of the dye released
135
/
0,6 R
m
0.6-C
0.4-
129
Elsevier BBM 00824
Quantitation of immobilized proteins * William S. Lewis 1 and Sheldon M. Schuster 2 1 Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE and 2 Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL, U.S.A.
(Received 8 February 1990) (Accepted 5 March 1990)
Summary A method for the quantitative determination of immobilized proteins based on the binding and subsequent elution of Coomassie Blue R is presented. Also presented is a method for the immobilization of proteins in solution by entrapment in polyacrylamide. These entrapped proteins are then available for use in the assay method presented. Other analytical procedures can also be performed on the entrapped proteins, either alone or in combination with the protein quantitation. The dye binding and elution method presented provides a sensitive and, in most applications, rapid method for the quantitative detection of immobilized proteins. Rather than immobilization being an obstacle to the assay method, this approach utilizes the advantages of immobilization for the removal of excess reagents. Application of this approach to several types of immobilized protein are presented.
Key words: Protein; Quantitation
Introduction Immobilization of proteins on or within solid-phase supports provides a convenient method for the manipulation of proteins and facilitates the removal of soluble
* This work was supported in part by a grant from Finnsugar Biochemicals, Inc.
Abbreviations: TEMED, N,N,N',N'-tetramethylethylenediamine; TCA, trichloroacetic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate; Coomassie Blue, Coomassie Brilliant Blue R-250; Bis, N, N '-methylenebisacrylamide.
Correspondence address: Sheldon M. Schuster, Interdisciplinary Center for Biotechnology Research, 1301 Fifield Hail, University of Florida, Gainesville, FL 32611, U.S.A. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
130 components from the immobilized protein. For most applications, the amount of protein attached to the support must be determined. Several methods have been developed for this purpose [1-3]. Since determinations are expressed as the amount of protein bound per unit volume of the support material, measurement of both the amount of protein in a sample and the volume of the support in the sample must be determined. Typically, the support is packed into a column and the volume of the packed column is measured. Then the amount of protein bound is determined either by the difference between the amount added and the amount remaining in the solution, following coupling or by one of several detection methods [1,2]. An alternative method is to pack the support into a cuvette and to measure the absorbance of the protein in the packed support [3]. These methods are tedious and require that relatively large volumes of the immobilized protein be available. In addition, little standardization of the pressure under which the support is packed is used, leading to large variations in the support volume determinatiofi. Here we report a method for the determination of the concentration of immobilized proteins. This method utilizes the binding and subsequent elution of a dye similar to several other systems which have previously been reported for proteins in solution [4-8]. The protein in the present method is immobilized during the staining, facilitating the removal of excess dye from the protein prior to elution of the protein-bound dye. Application of this method to proteins immobilized by several methods is reported. A method for the immobilization of proteins by addition of acrylamide to the solution and polymerization of the acrylamide is also presented. This method is applied, in conjunction with the method for the quantitation of immobilized proteins, for the removal of substances in the solution which interfere with the protein quantitation. A number of methods have been developed for the determination of total protein concentration in solutions [4-12]. All of these methods, however, exhibit some interference by reagents commonly found in the sample solutions [13-14]. Modifications of these procedures have resulted in numerous adaptations which eliminate one or a small group of these interferences [6,15]. A general method has yet to be developed for the assay of protein concentrations in samples free of interference by all of these substances. Since many samples for protein assays contain varying combinations of these substances, methods for the elimination of any one interfering substance are not generally applicable. Techniques for the removal of interfering reagents, such as dialysis, are inconvenient because of the small volumes and large number of samples involved and lack of quantitative recovery of the sample. Repeated precipitation of the protein with trichloroacteic acid is often used to remove interfering substances. This method is limited by low recovery of the protein and difficulty in dissolving the precipitated protein. This method can be utilized as an alternative to dialysis or other methods of reagent exchange. The proteins treated in this way can later be used for other analytical procedures, methods for many of which have been developed for acrylamide embedded proteins resulting from gel electrophoresis [16-18] and can be applied with little or no modification.
131 Materials and Methods Materials Methanol was obtained from Fisher (Fairlawn, N J) and distilled in glass. Coomassie Blue (C.I. 42660), Amido Black (C.I. 20470), Fast Green F C F (C.I. 42053), acrylamide, TEMED, .Bis and ammonium persulfate were Electrophoresis Grade obtained from Bio-Rad (Richmond, CA). Tissue culture plates (24 well) were from Sargent-Welch (Skokie, IL). HPLC grade pyridine was from Aldrich (Milwaukee, WI). Ammonium sulfate was from EM Science (Cherry Hill, N J). Sucrose was obtained from Schwarz/Mann Biotechnologies (Cleveland, OH), and chymotrypsin was from Worthington Biochemicals (Freehold, N J). All other reagents were obtained from Sigma Chemical Company (St. Louis, MO). Immobilization methods BSA was coupled to Sepharose 4B-CL by the CNBr method of Cuatrecasas [19]. The resin was activated in a single batch. The activated resin was then divided into approximately equal aliquots (10 ml packed resin each). Different amounts of BSA were added to the resin in a total volume of 10 ml of 0.1 M sodium phosphate buffer, p H 9.5. Following coupling at 4 ° C for 24 h, the remaining reactive sites on the resin were blocked by addition of ethanolamine to 1 M. The resin was removed by filtration and washed with 2-3 vol. of buffer. The filtrate and wash solutions were combined and the unbound protein was determined by measurement of the volume and absorbance of the combined solutions. Bound protein was calculated by the difference in absorbance between the protein solution added to the resin and the unbound protein. The volume of the substituted resin was determined by packing the resin into a graduated column. The concentration of the protein on the resin was determined by the amount of protein bound to the resin and the resulting volume of the resin. Immobilization of BSA on cellulose nitrate filters was accomplished by spotting Jifferent amounts of a stock BSA solution (1 m g / m l ) into each well of a Bio-Rad Dot-Blot apparatus. When all of the liquid was absorbed into the filter, a vacuum was applied and each well was washed with two 100 /~1 aliquots of 0.1 M sodium phosphate buffer, p H 6.8. Protein standards and standard protein assays Protein standards were made by carefully weighing out the desired amounts of protein and dissolving them in water. A single stock solution of each protein was used for all of the assay methods tested. Typically, stock solutions were used at a concentration of 1 mg/ml. Protein concentrations were determined by the methods of Lowry et al. [9] and Bradford [10] where indicated in the text. The method used for the Bradford determination was according to the standard assay protocol provided in the Bio-Rad Protein assay *. Absorbances of dye solutions were
* Bio-Rad protein assay, bulletin 1069 Bio-Rad Laboratories, Richmond, CA, U.S:A.
132 measured in disposable plastic semimicro cuvettes (4 x 10 m m with a 10 m m light path) obtained from Sarstadt using a Beckman DU-50 spectrophotometer. Radioactive resins were made by coupling of [14C]serine to a glycyl-Sepharose resin. Preparation of the resin was as described by Cuatrecasas for the coupling of alanine [19]. Coupling of the serine to the resin was accomplished by activation of the resin with D C C D to form the activated N-hydroxysuccinimide ester as described. For embedding of proteins in solution by entrapment in acrylamide, the following method was used. Protein samples containing the indicated amount of protein, and any other reagents as indicated, were diluted with water to a total volume of 50 #1 and placed in a 75 x 10 m m glass test tube. To each sample was added 25 #1 of a solution of 30% acrylamide * *, 0.8% Bis. Then 1.5 #1 of a freshly prepared solution of 10% a m m o n i u m persulfate was added to each sample. The samples were then mixed thoroughly and 1 #1 of T E M E D was added to each. The samples were left at room temperature for 10 min to allow complete polymerization. Gel electrophoresis of samples containing 0 - 1 0 #g of BSA were done using the Tris-borate gel system described by Sykes and Bailey [20]. Electrophoresis at 150 V was continued until the bromophenol Blue tracking dye reached the bottom of the gel slab.
Assay of immobilized proteins Sepharose immobilized proteins.
A portion of the resin suspension to be assayed is placed in a microcentrifuge tube. The resin is pelleted by centrifugation at 3000 r p m for 2 min. The resin is suspended in 1 ml of the staining solution which consists of 0.25% Coomassie Blue R, 30% methanol, and 10% acetic acid. After 10 min at room temperature, the sample is centrifuged for 3 min at 3000 rpm. The resin is suspended in 1 ml of the staining solution and incubated at room temperature for another 10 min. The sample is then centrifuged at 3000 rpm for 3 min and the supernatant fluid is removed. A small portion of the stained resin pellet, about 5 - 1 0 /xl, is carefully scooped out with a spatula and placed in another microfuge tube containing 1 ml of the destaining solution composed of 30% methanol and 7.5% acetic acid. After a few seconds of mixing to insure that the pellet is well suspended in the solution, the sample is centrifuged for 3 min at 5000 rpm. The supernatant fluid is decanted and saved for determination of the resin volume. The bound dye is eluted by suspending the pellet in 0.4 ml of a solution containing 1% SDS and 1% sodium bicarbonate. To this suspension is added 0.6 ml of methanol and the solution is mixed well to ensure thorough suspension of the resin. The resin is pelleted by centrifugation at 5000 r p m for 3 min and the supernatant fluid is decanted and saved for determination of the bound protein content of the resin sample. The amounts of dye in the supernatant fluids from the elution and destaining steps is determined by measurement of their absorbance at 595 nm. In the event
* * Percent solutions are expressed as weight/volume when the solute is a solid and volume/volume when the solute is a liquid at room temperature.
133 that the resin pellets become unstable during any of the decanting steps above, the preceding centrifugation step can be repeated to repack the pellet for complete removal of the supernatant fluid. Cellulose nitrate filter immobilized proteins. The filter containing the immobilized proteins is stained by incubation in a solution of 0.125% Coomassie Blue R, 30% methanol and 7.5% acetic.acid for 15 min with good mixing. The stained filter is then destained by several incubations with 30% methanol, 7.5% acetic acid until the solution remains colorless. The regions containing the protein to be assayed are then cut out with scissors and placed in separate wells of a 24-well tissue culture plate. The bound dye is eluted by incubation with 0.4 ml 1% SDS, 1% sodium bicarbonate, with good mixing for 30 min. The incubation is continued for an additional 60 min following addition of 0.6 ml methanol to each well. The amount of dye released is determined by measurement of the absorbance of the resulting solution at 595 nm. A crylamide embedded proteins. The gel containing the protein sample was removed from the test tube with a spatula and each sample was placed in a separate well of a 24-well tissue culture plate. The samples were incubated with the following sequence of reagents for at least 2 h each step. During incubation, mixing was accomplished by placing the plate on a benchtop shaker (Tektator V, S / P products, Evanston, IL). The wash solutions used were: - two washes with 10% trichloroacetic acid three washes with 10% acetic acid three washes with a solution of 5% methanol, 7.5% acetic acid two washes with a solution of 30% methanol, 7.5% acetic acid The samples were stained with 0.125% Coomassie Blue R in 35% methanol, 7.5% acetic acid for at least 6 h. The samples were destained with 30% methanol, 7.5% acetic acid using several changes in 2 ml portions. The samples were destained until blanks (samples containing no protein) appeared colorless. Typically the destaining required three changes of solution. When the samples were destained, the dye bound to the protein was eluted. In order to prevent evaporation during the elution step, a gasket was cut from a sheet of rubber to fit inside the lid of the tissue culture plate. The lid, with the gasket, was held in place by two rubber bands. As an additional precaution, the plate was sealed in a plastic reclosable sandwich bag. Except where noted, the following procedure was used for elution of the bound dye. The samples were incubated in 0.6 ml of a solution containing 1% SDS, 1% sodium bicarbonate for at least 2 h. Then 0.9 ml of methanol was added and incubation was continued for several more hours. This elution method was adapted from the protein assay described by Nakao et al. [6]. The combined elution solution (1.5 ml total volume) was collected and the absorbance of the eluted dye determined. Alternative methods for polyacrylamide embedded proteins. As described in Resuits, some samples were stained with Amido Black. For these samples, 1% Amido Black in 30% methanol, 7.5% acetic acid was used instead of the Coomassie Blue solution described above. All other steps remained the same. Alternative elution methods were also investigated. Two different methods were -
-
-
134 tested, elution with methanol and elution with pyridine. In samples eluted with methanol, the samples were eluted by incubating with 0.5 ml of 45% methanol, 10% acetic acid for 1 h. Then 0.5 ml of 80% methanol, 7.5% acetic acid was added to the well containing the gel and the first ehition solution. Following incubation for an additional hour, 0.5 ml of methanol was added and the incubation was continued for several more hours. This stepwise increase in the concentration of methanol was found to be necessary to insure complete elution of the dye from the interior of the gel. The resulting elution solution (1.5 ml total volume) was collected and the absorbance of the eluted dye was determined. Some samples were eluted from the gel pieces by incubation for several hours in 1.5 ml of a 25% pyridine solution. This method was adapted from Fenner et al. [21]. The elution solution was then removed and the absorbance of the eluted dye determined. Gel electrophoresis. Sections of the gel containing the protein bands or sections of varying size containing no protein were cut from the gel with a razor blade. Excess solution was removed by blotting the section dry with tissue paper and the section was weighed. The dye was then eluted from the gel as described previously except that the volumes were adjusted proportionally so that the total volume of the elution solution was 1.0 ml.
Results
Sepharose-immobilized proteins Application of the assay method to Sepharose-immobilized proteins involves the determination of both the volume of the resin in the sample a n d the amount of protein bound to the resin. The determination of the volume of resin used in the assay assumes that the volume of the bulk solution in and between the resin beads is proportional to the volume of resin present. The volume is then determined by the amount of dye removed from the resin in the destaining step. In order to test this assumption, a resin was synthesized to contain an immobilized 14C-labeled amino acid. A portion of this resin was packed in a 0.2 ml pipette which had been cut to produce a graduated column. The end of the pipette was covered by a piece of nylon mesh to allow the liquid portion of the resin suspension to pass through while retaining the resin. The volume of the packed resin was determined by the graduations of the pipette. This packed resin was then monitored to determine the radioactivity per ml of packed resin. Other portions of the resin were stained by the presented assay method and the amount of stain released in the destaining step was determined by its absorbance. The volume of resin in each sample was determined from the radioactivity present in the sample. The results of this experiment are shown in Fig: 1. The volume of dye released in the staining step is proportional to the volume of resin present. The amount of dye released in the elution step provides a measure of the amount of protein bound to the resin used in the sample. The amount of protein bound per unit volume of resin is proportional to the ratio of absorbances of the dye released
135
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m
0.6-C
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