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High-efficiency staining of proteins on different blot membranes
ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 338 (2005) 159–161 www.elsevier.com/locate/yabio
Notes & Tips
High-eYciency staining of proteins on diVerent blot membranes C.R. Yonan, P.T. Duong, F.N. Chang¤ Department of Biology, Temple University, Philadelphia, PA 19122, USA Received 18 June 2004 Available online 7 December 2004
Immunodetection of proteins on blot membranes, Wrst developed by Towbin et al. [1], is the method of choice for identifying speciWc proteins after electrophoresis. Due to its high resolution, immunoprobing of blots from twodimensional polyacrylamide gels has been used extensively as a means of localizing speciWc proteins among hundred or even thousands of separated proteins [2]. Three major types of membranes are used for protein blotting: nitrocellulose, nylon, and polyvinylidene diXuoride (PVDF).1 Typically, before immunodetection, a blot is subjected to a general protein stain such as Amido black 10B [3], Ponceau S [3], Coomassie brilliant blue R-250 [4], India ink [5], or Colloidal gold [2,6]. Of the protein stains, Amido black, Ponceau S, and Coomassie blue are the least sensitive, with a detection limit of approximately 50 ng protein. India ink has an intermediate sensitivity of detection and takes approximately 4 h to complete. Colloidal gold stain has the highest sensitivity on PVDF blot membrane (1–2 ng) but requires several hours to complete. To be eVective, most of the above protein staining procedures are carried out under acidic conditions (e.g., acetic acid) and/or organic solvents (e.g., methanol). The eYciency of staining also varied from membrane to membrane. For example, it has been reported that because of high staining background, nylon membrane is not compatible with all of the above dyes [7]. Furthermore, nitrocellulose membrane cannot be used when high concentrations of organic solvents such as methanol are used [7]. The use of acid and/or organic solvents on blots can also cause problems for the detection of labile antigenic activity, enzymatic activity, protein–protein interactions, and protein–ligand interactions [8]. *
Corresponding author. Fax: +1 215 204 6646. E-mail address: [email protected] (F.N. Chang). 1 Abbreviations used: PVDF, polyvinylidene diXuoride; BSA, bovine serum albumin; TTBS, Tris-buVered saline containing Tween 20. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.010
After visualization of protein spots on a blot, it is generally advantageous to remove the dyes from the blot so that immunoprobing can be carried out with the same membrane blot. Due to ease of removal, low-sensitivity dyes such as Ponceau S and Amido black 10B have been used extensively for this purpose [9]. Such a “reversible staining” process is diYcult to achieve with the more sensitive stains such as Colloidal gold, which requires rather harsh conditions to remove [9]. It is, therefore, highly desirable to develop a high-eYciency protein staining method with equal sensitivity toward all blot membranes that can be carried out under nondenaturing conditions. To our knowledge, reactive fabric dyes have not been reported for detecting proteins on membranes. While assessing various types of reactive fabric dye stains for their use as a general protein stain, we unexpectedly discovered that Reactive brown 10 (Fig. 1) stains proteins on blot membranes quickly with a sensitivity of detection comparable to that of Colloidal gold (1–2 ng). Proteins are visualized within a few seconds in the staining solution (0.05% in water). The Reactive brown 10 and other dyes were obtained from Sigma (St. Louis, MO, USA). Nitrocellulose and PVDF membranes were obtained from Bio-Rad (Hercules, CA, USA). Nylon membrane was obtained from Amersham (Buckinghamshire, UK). PVDF, nitrocellulose, and nylon membrane strips (1 £ 8 cm) were prepared. Each of the membrane strips was spotted with 1-l spots of bovine serum albumin (BSA) in a series of amounts consisting of 1, 2, 5, 50, 100, 500, and 1000 ng of protein. Due to the hydrophobic nature of the PVDF membrane, the protein solution for spotting of the PVDF membrane was mixed with an equal volume of -caprolactone before spotting. After drying of protein samples, the PVDF membrane was immersed in methanol (99.9%) brieXy before rinsing with distilled water for 15 min. Each membrane of each
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Notes & Tips / Anal. Biochem. 338 (2005) 159–161
Fig. 1. Structure of Reactive brown 10 fabric dye.
composition was then stained with one of a group of Wve staining compositions selected as follows. One membrane of each composition was stained using Ponceau S. In this procedure, the membrane was incubated in 0.1% (w/v) of Ponceau S stain in 5% acetic acid for 5 min. The membrane was then destained with deionized water. One membrane of each composition was stained using Amido black 10B. In this procedure, the membrane was incubated in a 0.1% (w/v) solution of Amido black dissolved in 10% (v/v) acetic acid and 30% (v/v) methanol. The membrane was incubated for 30 s, and the stain was removed. The membrane was destained with deionized water until the background was removed satisfactorily. One membrane of each composition was stained using Coomassie blue. In this procedure, the membrane was incubated in 0.125% Coomassie blue R-250 in 50% methanol and 10% acetic acid solution for 1 h. The stain was then removed, and the membrane was destained with 25% methanol until the background was removed. One membrane of each composition was stained using Colloidal gold. In this procedure, the membrane was washed three times for 20 min in Tris-buVered saline, pH 7.4, containing 0.05% Tween 20 (TTBS), followed by three 2-min washes in deionized water. The formulation of Colloidal gold was 5 ml of 1% gold chloride, 6 ml formamide, 5 ml Tween 20, and 3 ml of 0.2 M potassium hydroxide added dropwise, and the total volume was adjusted to 500 ml with water. The stain was allowed to stir vigorously overnight, and the pH was brought to 3.5 with formic acid. One membrane of each composition was stained using Reactive brown 10 by incubating the membranes with a 0.05% solution of Reactive brown 10 in distilled water for approximately 5–10 s. Destaining was carried out by washing the membrane in water for approximately 30 s. Fig. 2 shows the sensitivity of all the stain compositions on protein bound to PVDF membrane. It was noted that both Coomassie blue and Amido black 10B were diYcult to destain on PVDF membrane and that high background levels of the stain remained even after
extensive washing. Whereas the sensitivity of detection for Ponceau S, Amido black 10B, and Coomassie blue is approximately 50 ng, the sensitivity for Colloidal gold and Reactive brown is approximately 1 ng. Fig. 3 shows the sensitivity of all the stain compositions on protein bound to nitrocellulose membrane. Again, both Colloidal gold and Reactive brown are the most sensitive, with a detection limit of approximately 1 ng. Fig. 4 shows the sensitivity of all the stain compositions on protein bound to nylon membrane. None of the stain compositions, other than Reactive brown 10, was capable of eVecting protein detection. Reactive brown 10 yielded a reverse staining appearance but nonetheless gave the same sensitivity on nylon membrane (t1 ng) as on the other membrane compositions.
Fig. 2. Comparison of the sensitivities of various stains on PVDF membrane. BSA (1 mg/ml) was prepared as a stock solution, and a series of dilutions were spotted onto strips of PVDF membrane. Each membrane was then stained using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive brown 10.
Fig. 3. Comparison of the sensitivities of various stains on nitrocellulose membrane. A series of dilutions were spotted onto strips (as described previously) of nitrocellulose membrane. Each membrane was then stained using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive brown 10.
Notes & Tips / Anal. Biochem. 338 (2005) 159–161
161
brown 10 has been described. This procedure takes only a few seconds to stain and less than 1 min to destain. The sensitivity of protein staining at approximately 1 ng is comparable to that of the Colloidal gold method. Unlike the Colloidal gold method, this method also stains proteins on nylon membranes with the same eYciency as on other membranes. References Fig. 4. Comparison of the sensitivities of various stains on nylon membrane. A series of dilutions were spotted onto strips (as described previously) of nitrocellulose membrane. Each membrane was then stained using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive brown 10.
In the above studies, BSA was used as the standard protein. Essentially identical sensitivity toward Reactive brown 10 dye was observed with other proteins such as trypsin inhibitor and carbonic anhydrase. Unlike Colloidal gold staining, the Reactive brown 10 staining and destaining procedures were carried out in water and not under denaturing conditions (e.g., acidic and/or organic solvents). This will be of value for preservation of labile antigenic, binding, or catalytic activities when further characterization is required. In addition, unlike the Colloidal gold staining, the Reactive brown 10 stain is easily reversible; it can be completely removed by incubating under alkaline conditions (e.g., 0.1 N NaOH) for approximately 10 min so that the same blot can then be used for immunodetection. A simple procedure for fast and high-eYciency staining of proteins on various membranes using Reactive
[1] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350–4354. [2] M.J. Dunn, Detection of total proteins on Western blots of 2D polyacrylamide gels, Methods Mol. Biol. 112 (1999) 319–329. [3] K. Nakamura, T. Tanaka, A. Kuwahara, K. Takeo, Microassay for proteins on nitrocellulose Wlter using protein dye-staining procedure, Anal. Biochem. 148 (1985) 311–319. [4] W.N. Burnette, Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodiWed nitrocellulose and radiographic detection with antibody and radioiodinated protein A, Anal. Biochem. 112 (1981) 195–203. [5] K. Hancock, V.C. Tsang, India ink staining of proteins on nitrocellulose paper, Anal. Biochem. 133 (1983) 157–162. [6] M. Moeremans, G. Daneels, J. De Mey, Sensitive Colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes, Anal. Biochem. 145 (1985) 315–321. [7] M.G. Pluskal, M.B. Przekop, M.R. Kavonian, C. Vecoli, D.A. Hicks, Immobilon PVDF transfer membrane: a new membrane substrate for Western blotting of proteins, Biotechniques 4 (1986) 272–282. [8] F. Gentile, E. Bali, G. Pignalosa, Sensitivity and applications of the nondenaturing staining of proteins on polyvinylidene diXuoride membranes with Amido black 10B in water followed by destaining in water, Anal. Biochem. 245 (1997) 260–262. [9] B. Magi, L. Bini, B. Marzocchi, S. Liberatori, R. Raggiaschi, V. Pallini, ImmunoaYnity identiWcation of 2-DE separated proteins, Methods Mol. Biol. 112 (1999) 431–443.
Notes & Tips
High-eYciency staining of proteins on diVerent blot membranes C.R. Yonan, P.T. Duong, F.N. Chang¤ Department of Biology, Temple University, Philadelphia, PA 19122, USA Received 18 June 2004 Available online 7 December 2004
Immunodetection of proteins on blot membranes, Wrst developed by Towbin et al. [1], is the method of choice for identifying speciWc proteins after electrophoresis. Due to its high resolution, immunoprobing of blots from twodimensional polyacrylamide gels has been used extensively as a means of localizing speciWc proteins among hundred or even thousands of separated proteins [2]. Three major types of membranes are used for protein blotting: nitrocellulose, nylon, and polyvinylidene diXuoride (PVDF).1 Typically, before immunodetection, a blot is subjected to a general protein stain such as Amido black 10B [3], Ponceau S [3], Coomassie brilliant blue R-250 [4], India ink [5], or Colloidal gold [2,6]. Of the protein stains, Amido black, Ponceau S, and Coomassie blue are the least sensitive, with a detection limit of approximately 50 ng protein. India ink has an intermediate sensitivity of detection and takes approximately 4 h to complete. Colloidal gold stain has the highest sensitivity on PVDF blot membrane (1–2 ng) but requires several hours to complete. To be eVective, most of the above protein staining procedures are carried out under acidic conditions (e.g., acetic acid) and/or organic solvents (e.g., methanol). The eYciency of staining also varied from membrane to membrane. For example, it has been reported that because of high staining background, nylon membrane is not compatible with all of the above dyes [7]. Furthermore, nitrocellulose membrane cannot be used when high concentrations of organic solvents such as methanol are used [7]. The use of acid and/or organic solvents on blots can also cause problems for the detection of labile antigenic activity, enzymatic activity, protein–protein interactions, and protein–ligand interactions [8]. *
Corresponding author. Fax: +1 215 204 6646. E-mail address: [email protected] (F.N. Chang). 1 Abbreviations used: PVDF, polyvinylidene diXuoride; BSA, bovine serum albumin; TTBS, Tris-buVered saline containing Tween 20. 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.11.010
After visualization of protein spots on a blot, it is generally advantageous to remove the dyes from the blot so that immunoprobing can be carried out with the same membrane blot. Due to ease of removal, low-sensitivity dyes such as Ponceau S and Amido black 10B have been used extensively for this purpose [9]. Such a “reversible staining” process is diYcult to achieve with the more sensitive stains such as Colloidal gold, which requires rather harsh conditions to remove [9]. It is, therefore, highly desirable to develop a high-eYciency protein staining method with equal sensitivity toward all blot membranes that can be carried out under nondenaturing conditions. To our knowledge, reactive fabric dyes have not been reported for detecting proteins on membranes. While assessing various types of reactive fabric dye stains for their use as a general protein stain, we unexpectedly discovered that Reactive brown 10 (Fig. 1) stains proteins on blot membranes quickly with a sensitivity of detection comparable to that of Colloidal gold (1–2 ng). Proteins are visualized within a few seconds in the staining solution (0.05% in water). The Reactive brown 10 and other dyes were obtained from Sigma (St. Louis, MO, USA). Nitrocellulose and PVDF membranes were obtained from Bio-Rad (Hercules, CA, USA). Nylon membrane was obtained from Amersham (Buckinghamshire, UK). PVDF, nitrocellulose, and nylon membrane strips (1 £ 8 cm) were prepared. Each of the membrane strips was spotted with 1-l spots of bovine serum albumin (BSA) in a series of amounts consisting of 1, 2, 5, 50, 100, 500, and 1000 ng of protein. Due to the hydrophobic nature of the PVDF membrane, the protein solution for spotting of the PVDF membrane was mixed with an equal volume of -caprolactone before spotting. After drying of protein samples, the PVDF membrane was immersed in methanol (99.9%) brieXy before rinsing with distilled water for 15 min. Each membrane of each
160
Notes & Tips / Anal. Biochem. 338 (2005) 159–161
Fig. 1. Structure of Reactive brown 10 fabric dye.
composition was then stained with one of a group of Wve staining compositions selected as follows. One membrane of each composition was stained using Ponceau S. In this procedure, the membrane was incubated in 0.1% (w/v) of Ponceau S stain in 5% acetic acid for 5 min. The membrane was then destained with deionized water. One membrane of each composition was stained using Amido black 10B. In this procedure, the membrane was incubated in a 0.1% (w/v) solution of Amido black dissolved in 10% (v/v) acetic acid and 30% (v/v) methanol. The membrane was incubated for 30 s, and the stain was removed. The membrane was destained with deionized water until the background was removed satisfactorily. One membrane of each composition was stained using Coomassie blue. In this procedure, the membrane was incubated in 0.125% Coomassie blue R-250 in 50% methanol and 10% acetic acid solution for 1 h. The stain was then removed, and the membrane was destained with 25% methanol until the background was removed. One membrane of each composition was stained using Colloidal gold. In this procedure, the membrane was washed three times for 20 min in Tris-buVered saline, pH 7.4, containing 0.05% Tween 20 (TTBS), followed by three 2-min washes in deionized water. The formulation of Colloidal gold was 5 ml of 1% gold chloride, 6 ml formamide, 5 ml Tween 20, and 3 ml of 0.2 M potassium hydroxide added dropwise, and the total volume was adjusted to 500 ml with water. The stain was allowed to stir vigorously overnight, and the pH was brought to 3.5 with formic acid. One membrane of each composition was stained using Reactive brown 10 by incubating the membranes with a 0.05% solution of Reactive brown 10 in distilled water for approximately 5–10 s. Destaining was carried out by washing the membrane in water for approximately 30 s. Fig. 2 shows the sensitivity of all the stain compositions on protein bound to PVDF membrane. It was noted that both Coomassie blue and Amido black 10B were diYcult to destain on PVDF membrane and that high background levels of the stain remained even after
extensive washing. Whereas the sensitivity of detection for Ponceau S, Amido black 10B, and Coomassie blue is approximately 50 ng, the sensitivity for Colloidal gold and Reactive brown is approximately 1 ng. Fig. 3 shows the sensitivity of all the stain compositions on protein bound to nitrocellulose membrane. Again, both Colloidal gold and Reactive brown are the most sensitive, with a detection limit of approximately 1 ng. Fig. 4 shows the sensitivity of all the stain compositions on protein bound to nylon membrane. None of the stain compositions, other than Reactive brown 10, was capable of eVecting protein detection. Reactive brown 10 yielded a reverse staining appearance but nonetheless gave the same sensitivity on nylon membrane (t1 ng) as on the other membrane compositions.
Fig. 2. Comparison of the sensitivities of various stains on PVDF membrane. BSA (1 mg/ml) was prepared as a stock solution, and a series of dilutions were spotted onto strips of PVDF membrane. Each membrane was then stained using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive brown 10.
Fig. 3. Comparison of the sensitivities of various stains on nitrocellulose membrane. A series of dilutions were spotted onto strips (as described previously) of nitrocellulose membrane. Each membrane was then stained using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive brown 10.
Notes & Tips / Anal. Biochem. 338 (2005) 159–161
161
brown 10 has been described. This procedure takes only a few seconds to stain and less than 1 min to destain. The sensitivity of protein staining at approximately 1 ng is comparable to that of the Colloidal gold method. Unlike the Colloidal gold method, this method also stains proteins on nylon membranes with the same eYciency as on other membranes. References Fig. 4. Comparison of the sensitivities of various stains on nylon membrane. A series of dilutions were spotted onto strips (as described previously) of nitrocellulose membrane. Each membrane was then stained using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive brown 10.
In the above studies, BSA was used as the standard protein. Essentially identical sensitivity toward Reactive brown 10 dye was observed with other proteins such as trypsin inhibitor and carbonic anhydrase. Unlike Colloidal gold staining, the Reactive brown 10 staining and destaining procedures were carried out in water and not under denaturing conditions (e.g., acidic and/or organic solvents). This will be of value for preservation of labile antigenic, binding, or catalytic activities when further characterization is required. In addition, unlike the Colloidal gold staining, the Reactive brown 10 stain is easily reversible; it can be completely removed by incubating under alkaline conditions (e.g., 0.1 N NaOH) for approximately 10 min so that the same blot can then be used for immunodetection. A simple procedure for fast and high-eYciency staining of proteins on various membranes using Reactive
[1] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) 4350–4354. [2] M.J. Dunn, Detection of total proteins on Western blots of 2D polyacrylamide gels, Methods Mol. Biol. 112 (1999) 319–329. [3] K. Nakamura, T. Tanaka, A. Kuwahara, K. Takeo, Microassay for proteins on nitrocellulose Wlter using protein dye-staining procedure, Anal. Biochem. 148 (1985) 311–319. [4] W.N. Burnette, Western blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodiWed nitrocellulose and radiographic detection with antibody and radioiodinated protein A, Anal. Biochem. 112 (1981) 195–203. [5] K. Hancock, V.C. Tsang, India ink staining of proteins on nitrocellulose paper, Anal. Biochem. 133 (1983) 157–162. [6] M. Moeremans, G. Daneels, J. De Mey, Sensitive Colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes, Anal. Biochem. 145 (1985) 315–321. [7] M.G. Pluskal, M.B. Przekop, M.R. Kavonian, C. Vecoli, D.A. Hicks, Immobilon PVDF transfer membrane: a new membrane substrate for Western blotting of proteins, Biotechniques 4 (1986) 272–282. [8] F. Gentile, E. Bali, G. Pignalosa, Sensitivity and applications of the nondenaturing staining of proteins on polyvinylidene diXuoride membranes with Amido black 10B in water followed by destaining in water, Anal. Biochem. 245 (1997) 260–262. [9] B. Magi, L. Bini, B. Marzocchi, S. Liberatori, R. Raggiaschi, V. Pallini, ImmunoaYnity identiWcation of 2-DE separated proteins, Methods Mol. Biol. 112 (1999) 431–443.