High-Eyciency 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 After visualization of protein spots on a blot, it is gen- developed by Towbin et al. [1], is the method of choice for erally advantageous to remove the dyes from the blot so identifying speciWc proteins after electrophoresis. Due to that immunoprobing can be carried out with the same its high resolution, immunoprobing of blots from two- membrane blot. Due to ease of removal, low-sensitivity dimensional polyacrylamide gels has been used exten- dyes such as Ponceau S and Amido black 10B have been sively as a means of localizing speciWc proteins among used extensively for this purpose [9]. Such a “reversible hundred or even thousands of separated proteins [2]. staining” process is diYcult to achieve with the more Three major types of membranes are used for protein sensitive stains such as Colloidal gold, which requires blotting: nitrocellulose, nylon, and polyvinylidene diXuo- rather harsh conditions to remove [9]. It is, therefore, ride (PVDF).1 Typically, before immunodetection, a blot highly desirable to develop a high-eYciency protein is subjected to a general protein stain such as Amido black staining method with equal sensitivity toward all blot 10B [3], Ponceau S [3], Coomassie brilliant blue R-250 [4], membranes that can be carried out under nondenaturing India ink [5], or Colloidal gold [2,6]. Of the protein stains, conditions. To our knowledge, reactive fabric dyes have Amido black, Ponceau S, and Coomassie blue are the not been reported for detecting proteins on membranes. least sensitive, with a detection limit of approximately While assessing various types of reactive fabric dye 50 ng protein. India ink has an intermediate sensitivity of stains for their use as a general protein stain, we unex- detection and takes approximately 4 h to complete. Colloi- pectedly discovered that Reactive brown 10 (Fig. 1) dal gold stain has the highest sensitivity on PVDF blot stains proteins on blot membranes quickly with a sensi- membrane (1–2 ng) but requires several hours to complete. tivity of detection comparable to that of Colloidal gold To be eVective, most of the above protein staining proce- (1–2 ng). Proteins are visualized within a few seconds in dures are carried out under acidic conditions (e.g., acetic the staining solution (0.05% in water). acid) and/or organic solvents (e.g., methanol). The The Reactive brown 10 and other dyes were obtained eYciency of staining also varied from membrane to mem- from Sigma (St. Louis, MO, USA). Nitrocellulose and brane. For example, it has been reported that because of PVDF membranes were obtained from Bio-Rad (Hercu- high staining background, nylon membrane is not com- les, CA, USA). Nylon membrane was obtained from patible with all of the above dyes [7]. Furthermore, nitro- Amersham (Buckinghamshire, UK). cellulose membrane cannot be used when high PVDF, nitrocellulose, and nylon membrane strips concentrations of organic solvents such as methanol are (1 £ 8 cm) were prepared. Each of the membrane strips used [7]. The use of acid and/or organic solvents on blots was spotted with 1-l spots of bovine serum albumin can also cause problems for the detection of labile anti- (BSA) in a series of amounts consisting of 1, 2, 5, 50, 100, genic activity, enzymatic activity, protein–protein interac- 500, and 1000 ng of protein. Due to the hydrophobic tions, and protein–ligand interactions [8]. 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 * Corresponding author. Fax: +1 215 204 6646. E-mail address: [email protected] (F.N. Chang). drying of protein samples, the PVDF membrane was 1 Abbreviations used: PVDF, polyvinylidene diXuoride; BSA, bovine immersed in methanol (99.9%) brieXy before rinsing with serum albumin; TTBS, Tris-buVered saline containing Tween 20. distilled water for 15 min. Each membrane of each

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 1ng. Fig. 4 shows the sensitivity of all the stain composi- Fig. 1. Structure of Reactive brown 10 fabric dye. tions on protein bound to nylon membrane. None of the stain compositions, other than Reactive brown 10, was composition was then stained with one of a group of Wve capable of eVecting protein detection. Reactive brown 10 staining compositions selected as follows. yielded a reverse staining appearance but nonetheless One membrane of each composition was stained gave the same sensitivity on nylon membrane (t1 ng) as using Ponceau S. In this procedure, the membrane was on the other membrane compositions. 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 satis- factorily. 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% Fig. 2. Comparison of the sensitivities of various stains on PVDF methanol and 10% acetic acid solution for 1 h. The membrane. BSA (1 mg/ml) was prepared as a stock solution, and a stain was then removed, and the membrane was series of dilutions were spotted onto strips of PVDF membrane. Each destained with 25% methanol until the background was membrane was then stained using a diVerent staining method. Lane 1: removed. membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido black 10B. Lane 3: membrane strip stained One membrane of each composition was stained with 0.125% Coomassie blue. Lane 4: membrane strip stained with using Colloidal gold. In this procedure, the membrane Colloidal gold. Lane 5: membrane strip stained with 0.05% Reactive was washed three times for 20 min in Tris-buVered brown 10. saline, pH 7.4, containing 0.05% Tween 20 (TTBS), followed by three 2-min washes in deionized water. The for- mulation of Colloidal gold was 5 ml of 1% gold chloride, 6 ml formamide, 5 ml Tween 20, and 3 ml of 0.2 M potas- sium 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 approxi- Fig. 3. Comparison of the sensitivities of various stains on nitrocellu- mately 30 s. lose membrane. A series of dilutions were spotted onto strips (as Fig. 2 shows the sensitivity of all the stain composi- described previously) of nitrocellulose membrane. Each membrane was tions on protein bound to PVDF membrane. It was then stained using a diVerent staining method. Lane 1: membrane strip noted that both Coomassie blue and Amido black 10B stained with 0.1% Ponceau S. Lane 2: membrane strip stained with Y 0.1% Amido black 10B. Lane 3: membrane strip stained with 0.125% were di cult to destain on PVDF membrane and that Coomassie blue. Lane 4: membrane strip stained with Colloidal gold. high background levels of the stain remained even after 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 mem- [1] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of brane. A series of dilutions were spotted onto strips (as described pre- proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. USA 76 (1979) viously) of nitrocellulose membrane. Each membrane was then stained 4350–4354. using a diVerent staining method. Lane 1: membrane strip stained with 0.1% Ponceau S. Lane 2: membrane strip stained with 0.1% Amido [2] M.J. Dunn, Detection of total proteins on Western blots of 2D polyacrylamide gels, Methods Mol. Biol. 112 (1999) 319–329. black 10B. Lane 3: membrane strip stained with 0.125% Coomassie [3] K. Nakamura, T. Tanaka, A. Kuwahara, K. Takeo, Microassay blue. Lane 4: membrane strip stained with Colloidal gold. Lane 5: for proteins on nitrocellulose Wlter using protein dye-staining pro- membrane strip stained with 0.05% Reactive brown 10. cedure, Anal. Biochem. 148 (1985) 311–319. [4] W.N. Burnette, Western blotting: electrophoretic transfer of pro- In the above studies, BSA was used as the standard teins from sodium dodecyl sulfate–polyacrylamide gels to protein. Essentially identical sensitivity toward Reactive unmodiWed nitrocellulose and radiographic detection with anti- brown 10 dye was observed with other proteins such as body and radioiodinated protein A, Anal. Biochem. 112 (1981) trypsin inhibitor and carbonic anhydrase. 195–203. Unlike Colloidal gold staining, the Reactive brown 10 [5] K. Hancock, V.C. Tsang, India ink staining of proteins on nitrocellulose paper, Anal. Biochem. 133 (1983) 157–162. staining and destaining procedures were carried out in [6] M. Moeremans, G. Daneels, J. De Mey, Sensitive Colloidal metal water and not under denaturing conditions (e.g., acidic (gold or silver) staining of protein blots on nitrocellulose mem- and/or organic solvents). This will be of value for preser- branes, Anal. Biochem. 145 (1985) 315–321. vation of labile antigenic, binding, or catalytic activities [7] M.G. Pluskal, M.B. Przekop, M.R. Kavonian, C. Vecoli, D.A. when further characterization is required. In addition, Hicks, Immobilon PVDF transfer membrane: a new membrane substrate for Western blotting of proteins, Biotechniques 4 (1986) unlike the Colloidal gold staining, the Reactive brown 10 272–282. stain is easily reversible; it can be completely removed by [8] F. Gentile, E. Bali, G. Pignalosa, Sensitivity and applications of incubating under alkaline conditions (e.g., 0.1 N NaOH) the nondenaturing staining of proteins on polyvinylidene diXuo- for approximately 10 min so that the same blot can then ride membranes with Amido black 10B in water followed by be used for immunodetection. destaining in water, Anal. Biochem. 245 (1997) 260–262. Y [9] B. Magi, L. Bini, B. Marzocchi, S. Liberatori, R. Raggiaschi, V. A simple procedure for fast and high-e ciency stain- Pallini, ImmunoaYnity identiWcation of 2-DE separated proteins, ing of proteins on various membranes using Reactive Methods Mol. Biol. 112 (1999) 431–443.