Microwave-Enhanced Ink Staining for Fast and Sensitive Protein

Microwave-Enhanced Ink Staining for Fast and Sensitive Protein Quantification in Proteomic Studies Xue-Ping Wu, Yong-Sheng Cheng, and Jin-Yuan Liu* Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China Received June 12, 2006

Abstract: A novel microwave-enhanced ink staining method was developed for rapid and sensitive estimation of protein content in sample buffers containing chaotropes, dyes, detergents, and reducing agents. Dye-based BlueBlack ink was used to quantitatively visualize proteins spotted on a nitrocellulose membrane. The total staining time was greatly reduced to 3 min by brief exposure to microwave radiation. The stained membrane was washed with distilled water, baked in a microwave oven for complete desiccation, transparentized with mineral oil, and documented by a desktop scanner or densitometer. Only 1 µL of protein sample (protein solubilized in SDSPAGE sample buffer or IEF rehydration buffer) was used for protein spotting. The novel solid-phase protein assay gives a 500-fold dynamic range from 19.5 to 10000 ng/µL and can be scaled up for high-throughput protein quantification analysis. The fast, sensitive and low-cost microwave-enhanced ink staining procedure is ideal for protein quantification in proteomic analysis. Keywords: microwave • ink staining • protein quantification • IEF rehydration buffer • SDS-PAGE sample buffer • quantitative proteomics

Introduction Despite the recent development of sophisticated methods for proteomics, obsolescent protein quantification is still necessary at the very beginning of sample workflow. The total protein content in the sample buffers (SDS-PAGE sample buffer and IEF rehydration buffer) has to be accurately controlled for adequate protein loading amounts and reliable comparison among different samples in regard to diversified protein expression. It is an emerging demand that protein quantification methods optimized for proteomic analysis should be rapid, sensitive, high-throughput, and have a low consumption of limited samples. Common spectrophotometric methods, such as the Bradford,1 BCA,2 and Lowry3 methods, have been extensively used for protein quantification. However, these insolution methods are restricted by the presence of interfering substances including CHAPS, SDS, DTT, thiourea, carrier ampholytes, and high-concentration chaotropes. Also, the * Corresponding author. Tel: +86-10-62772243; fax: +86-10-62772243; e-mail: [email protected]. 10.1021/pr0602848 CCC: $37.00

 2007 American Chemical Society

tracking dye-like bromophenol blue will influence the readout, especially in the Bradford assay. Some commercial kits that claim to be compatible with such interfering substances, such as the RC DC protein assay kit from Bio-Rad (Bio-Rad Laboratories, Hercules, CA) and the 2-D Quant kit from Amersham (Amersham Biosciences, Piscataway, NJ), usually involve protein precipitation, which consumes a large amount of sample and causes severe protein loss in the case of a low protein concentration. A solid-phase protein assay provides a good alternative.4-14 With the protein immobilized on a solid surface such as a nitrocellulose membrane or a PVDF membrane, interfering substances could be washed away. Thus, appropriate staining techniques are required to detect proteins in the nanogram to microgram range. Conventional on-membrane protein staining methods include colloidal gold staining, colloidal silver staining, Coomassie blue staining, Ponceau S staining, pyrogallol redmolybdate staining, India ink staining, and double-metal chelate (DMC) staining.5,10,11,14-18 Many of these methods, however, involve time-consuming procedures and multiple reagents. Recent fluorescence-based protein quantification methods have become well-established for high-throughput applications.8,13 Furthermore, limited protein samples as little as 1 µL are required in these sensitive on-membrane protein detection methods. Nevertheless, fluorescence-based methods necessitate costly fluorescent probes and expensive fluorescence laser scanners, UV illumination devices, or 96-well fluorescence plate readers. Microwave-enhanced chemistry has been widely employed in life science research.19-22 Microwave treatment is a good alternative to conventional heating. Nowadays, a microwave oven is the most common apparatus used for the preparation of agarose gel. Furthermore, microwave radiation can also accelerate some chemical and physical processes. Recently, protein digestion by trypsin and chemical reagents has also been largely accelerated by microwave radiation.19,20 Additionally, microwave-enhanced acidic protein digestion performed under careful control has been used for de novo peptide sequencing.21,22 Here, a novel microwave-assisted ink staining assay was developed for protein quantification in electrophoresis sample buffers. Ordinary dye-containing Blue-Black ink was applied for sensitive background-free protein staining on a nitrocellulose membrane. Microwave radiation largely reduced the staining time to 3 min. The microwave-desiccated membrane was then documented by a desktop scanner or densitometer, Journal of Proteome Research 2007, 6, 387-391

387

Published on Web 12/08/2006

technical notes

Protein Quantification in Proteomic Studies

and the Image J (http://rsb.info.nih.gov/ij/) software was used to evaluate scanned images. This low-cost protein assay could be easily scaled up for high-throughput applications by dotting unlimited protein samples on sizable nitrocellulose membranes and requires only 1 µL sample per spot. The assay offers a 500fold dynamic range that is comparable with common fluorescence-based detection methods. The current solid-phase microwave-enhanced protein assay is rapid, simple, sensitive, lowcost, reproducible, and high-throughput and provides a good alternative to expensive fluorescence-based assays.

Materials and Methods Materials. BSA, γ-globin, Con A, myoglobin, and ovalbumin were purchased from Sigma-Aldrich (Sigma-Aldrich Corp., St. Louis, MO). Apo-transferrin was purchased from Merck (Merck & Co., Inc., Whitehouse Station, NJ). BSA was dissolved in SDSPAGE sample buffer (100 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.1% bromophenol blue) and IEF rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT, and 0.5% IPG 3-10NL buffer), respectively, for dynamic range establishment. BSA, γ-globin, ConA, myoglobin, ovalbumin, and apo-transferrin were solubilized in IEF rehydration buffer for verification of protein-to-protein variability. Various kinds of ink, including Ostrich Blue-Black ink (Model 203), Boss Blue-Black ink (Model 883), Boss Black ink (Model 881), Hero Blue-Black ink (Model 232), Oriental Red ink (Model 8201), Oriental Blue-Black ink (Model 8202), and Oriental Blue ink (Model 8203) were obtained from a local art store (Haidian District, Beijing, China). The nitrocellulose membrane Immobilon-P was from Millipore (Millipore, Billerica, MA). A domestic microwave oven with a 900 W output was obtained from Galanz (Galanz Enterprises Group Co., GuangDong, China). A PowerLook 2100 XL desktop scanner was purchased from Umax (Maxium Technologies Inc., Shanghai, China). All other reagents were of the highest quality available. Preparation of Membrane and Staining Solution. Protein samples with concentrations ranging from 19.5 to 10000 ng/ µL were spotted on a nitrocellulose membrane as 1 µL dots. The spotted membrane was air-dried at room temperature. The dot-blotted membrane was then washed with distilled water 3 times within 5 min and readied for microwave ink staining. The ink staining solution used was 1% ink and 0.03% Tween20 in PBS. On-Membrane Protein Assay. Sets of protein standards were prepared by 2-fold gradient dilution of 10000 ng/µL protein samples, which were initially controlled at 10000 ng/µL by measuring dry protein powder gravimetrically and dissolving in sample buffers. Standard curves were acquired by spotting protein standards dissolved in SDS-PAGE sample buffer and IEF rehydration buffer on a nitrocellulose membrane. A 900 W domestic microwave oven from Galanz was utilized to provide microwave radiation to enhance the ink staining. Protein samples were spotted on nitrocellulose membrane as a 1 µL aliquot. Then, the membrane was air-dried and washed with distilled water 3 times in 5 min and was ready for microwaveassisted ink staining. Most interfering substances were washed away at this stage. Microwave-assisted ink staining was performed under microwave radiation for 3 min. After brief washing with distilled water, the stained membrane was desiccated by microwave radiation for 5 min and then rinsed with mineral oil or a cover fluid (Amersham Biosciences) for complete semi-transparentness. The semi-translucent mem388

Journal of Proteome Research • Vol. 6, No. 1, 2007

Figure 1. Comparison between microwave-enhanced staining and conventional staining. All membranes were stained in a Petri dish with an ink staining solution. Microwave staining was performed under 900 W microwave radiation treatment for 1, 2, 3, and 5 min, respectively. Conventional staining was accomplished by incubation at room temperature for 2 h and overnight separately.

branes were ready for documentation by a desktop scanner or densitometer. Image Analysis. Protein spots on the membrane were evaluated with Image J software (formally NIH image software), which is free software developed by Wayne Rasband at NIH. The pixel intensity value was calibrated with a standard optical density tablet and then converted into calibrated optical density value that is proportional to the amount of protein in the spot. Standard curves were plotted with the protein amount against the average absorbance unit in each spot.

Results and Discussion Blue-Black Ink Can Be a Sensitive Probe for Protein Immobilized on Membrane. Several dye-based inks and India ink were investigated for on-membrane protein staining. Four BSA samples in rehydration buffer with concentrations of 10, 100, 1000, and 10000 ng/µL were spotted on a membrane in 1 µL aliquots. Of a number of dye-based inks, only Blue-Black ink from all brands (including Ostrich Blue-Black ink, Hero Blue-Black ink, Boss Blue-Black ink, and Oriental Blue-Black ink) gave acceptable staining sensitivity, while others had a high background (like Boss Black ink) and/or low sensitivity (like Oriental Red ink and Oriental Blue ink) (data not shown). Dyebased ink stained proteins on the membrane showed the color of the corresponding ink, which could be the result of proteinspecific dye binding. India ink also did not yield practicable staining intensity (data not shown). Moreover, Ostrich BlueBlack ink demonstrated the highest sensitivity in comparison to Blue-Black inks of other brands, and it was used in a subsequent protein assay. Microwave Radiation Enhances the Ink Staining. The staining time with conventional India ink or dye-based ink was 2 h or overnight at room temperature. In addition, protein spots on the membrane were visible after incubation at room temperature for 15 min. Microwave radiation shortened the total staining time to just 3 min. Moreover, spotted proteins on the membrane were visible after 20 s of microwave treatment (Figure 1). It is possible that acceleration of ink staining within 5 min is the result of heating and non-heating effects

technical notes of microwave radiation, which synergistically increased the rate of protein specific dye binding. The microwave staining time was optimized to 3 min for sufficient sensitivity and satisfactory maintenance of the spot shape. The 5 min of microwaveradiation treatment caused spot deformation. In addition, microwave treatment intensified the staining of low-concentration protein spots in comparison with conventional room temperature incubation. Microwave-Accelerated Membrane Documentation. Membranes loaded with stained proteins should be air-dried, transparentized by mineral oil, and documented by a desktop scanner or densitometer. Usually, air-drying for 30 min or more is adequate for the complete desiccation of the membrane. The complete dry membrane can then be easily transparentized by mineral oil. Microwave radiation can also be utilized to accelerate the membrane documentation by shortening the time of membrane desiccation. A 5 min treatment of microwave radiation at a 900 W output is enough for complete desiccation of a stained membrane. After 5 min microwave desiccation and transparentization by mineral oil, the semi-translucent membrane is ready for documentation by transimissive scanning using a desktop scanner or densitometer. The transparentization with mineral oil is used to achieve the translucency of background and brightness of spots (stained spots were not transparent). Other membrane documentation methods, including reflective scanning of dry and wet membrane without transparentization, have also been investigated. Both reflective scanning methods did not give good images of stained spots (data not shown). Using the membranes lubricated with mineral oil, proteins can be detected with greater sensitivity in comparison to the membranes without lubrication. Furthermore, the flatness of the membrane surface could be a critical condition for successful reflective scanning, while it does not influence transmissive scanning of the oil-transparentized membrane to a certain extent. The color of the spotted proteins on the transparentized membrane was stable in mineral oil at room temperature. The dye-protein complex was so stable in mineral oil that the intensity and shape of the protein spots did not change after storage for several weeks. High-Throughput Protein Assays and Reuse of the Staining Solution. Present protein assays can be easily adapted for highthroughput protein quantification analysis. An unlimited number of protein samples can be spotted onto sizable nitrocellulose membranes, and then these spotted membranes can be stained in a single Petri dish at the same time. Protein samples can also be spotted on the nitrocellulose membrane one time or spotted separately several times. It would be convenient for protein samples prepared at different times to be spotted at different times onto the same membrane and stained simultaneously. Moreover, the staining solution can be reused many times. All membranes spotted with protein samples were stained using the same staining solution throughout our experiments. The dye capacity and staining intensity of the ink solution were not noticeably changed. However, the volume of the staining solution could be decreased by microwave radiation due to the evaporation of water. To overcome this problem, an adequate volume of distilled water was required to restore the original volume of the staining solution. Taken together, the current protein assay can be adapted to highthroughput protein estimations in a very economical way. Dynamic Range of Microwave Staining. For estimation of the protein content in an electrophoresis sample, BSA was dissolved in SDS-PAGE sample buffer or IEF rehydration buffer

Wu et al.

Figure 2. Standard curve determination for protein samples in rehydration buffer. Protein concentrations of standards were extended from 19.5 to 10000 ng/µL. Ostrich Blue-Black ink was used in the protein assay. The standard curve was plotted as the average optical density of each spot vs corresponding proteinloading amount. The inset in panel A is the shortened range of the standard curve extending from 19.5 to 1250 ng/µL. Panel B shows the scanned image of stained protein standards immobilized on a nitrocellulose membrane. Protein standards dissolved in rehydration buffer were dot-spotted on the membrane as 1 µL aliquots in triplicate and then air-dried. Staining was achieved by a 3 min treatment with microwave radiation.

and serially diluted as protein standards. Figures 2B and 3B show the scanned images of BSA standard curves in the sample buffers. Each protein concentration was spotted in triplicate onto the membrane and stained with 3 min of microwave radiation. The CV for each protein concentration was less than 3%. The BSA standard curves in sample buffers are shown in Figures 2A and 3A. The dynamic range of BSA in both buffers was 19.5s10 000 ng/µL, while the linear ranges for SDS-PAGE buffer and IEF rehydration buffer were 39-1000 and 19.5-2500 ng/µL, respectively. These linear ranges for sample buffers are sufficient to cover the common-use concentration range of protein samples that can be directly submitted for SDS-PAGE analysis and IEF separation. The linear range of BSA standards in SDS-PAGE buffer is shorter than that in IEF rehydration buffer, which could be due to the interfering effect of SDS on protein immobilization on the nitrocellulose membrane. The nonlinear relationship between protein amount and staining intensity outside the linear range could be the result of unsaturated dye binding. When the protein loading amount excel 1 µg, the crowdedness of protein immobilization reduced the accessibility of the dye. The unsaturated dye binding reduced the relative staining intensity of the spots with high protein loading. The reduction in staining intensity became greater as the protein loading amout was increased. Protein-to-Protein Variability. Six different proteins with diverse molecular weight, pI, and post-translational modifications were dissolved in IEF rehydration buffer and spotted onto a nitrocellulose membrane in triplicate. Figure 4 shows a summary of protein-to-protein variability verifications. The largest variation was caused by Con A, which is a glycoprotein (UniPort accession number P02866) has only one glycosylation site at Asn152. Con A could be potentially glycosylated with N-linked GlcNAc. The relative staining intensity of Con A is much lower than the average of all six proteins, even if the weight contribution of the sugar group has been corrected. The possible reason for reduced staining intensity as compared to the average could be the lower dye binding capacity or weaker immobilization of glycoproteins on the nitrocellulose memJournal of Proteome Research • Vol. 6, No. 1, 2007 389

technical notes

Protein Quantification in Proteomic Studies

Figure 3. Standard curve determination for protein samples in SDS sample buffer. Protein concentrations of standards were extended from 19.5 to 10000 ng/µL. The standard curve was plotted as average optical density of each spot vs corresponding protein-loading amount. The inset in panel A is the shortened range of the standard curve extending from 19.5 to 1250 ng/µL. Panel B shows the scanned image of stained protein standards immobilized on a nitrocellulose membrane. Protein standards dissolved in SDS sample buffer were dot-spotted on the membrane as 1 µL aliquots in triplicate and then air-dried. Staining was achieved by a 3 min treatment with microwave radiation.

microwave-enhanced ink staining produces background-free and sensitive protein staining on a nitrocellulose membrane, which is comparable with fluorescence-based solid-phase protein quantification methods. The entire protein content analysis could be finished within 15 min. Only low-priced and commonly used reagents and equipments were involved in the whole experimental procedure. Combining the advantages of solid-phase protein assay and microwave-enhanced ink staining, the protein quantification method reported here could have many potential applications, such as protein quantification in various lysis buffers. Abbreviations: BSA, bovine serum albumin; SDS-PAGE, SDS polyacrylamide gel electrophoresis; IEF, isoelectric focusing; BCA, bicinchoninic acid; CHAPS, 3-3-(cholamidopropyl)-dimethyl-ammonio-1-propane sulfonate; DTT, dithiothreitol; PVDF, polyvinylidene fluoride; CV, coefficient of variation. Figure 4. Protein-to-protein variability was evaluated from microwave-enhanced staining of six proteins dissolved in IEF rehydration buffer at a concentration of 10 mg/mL. The 1 µL aliquots of each proten were dot-spotted onto a nitrocellulose membrane in triplicate.

brane. Further experiments on the mechanism of microwaveassisted ink staining could shed light on the low signal intensity of Con A. The CVs of individual proteins were no more than 3%, and the CV among these six proteins was calculated to be 30.7%, as compared with a CV of 15.4% in the absence of Con A. This variability is comparable to those of common protein staining methods including Coomassie Blue staining and the BCA method. γ-Globin, myoglobin, and apo-transferrin could be better protein standards than BSA, for their relative staining intensities are closer to the average.

Conclusion Here, we have introduced a novel solid-phase protein assay with low-cost, rapid, simple, sensitive, reproducible, and highthroughput advantages. The newly introduced ink staining is dye-based and microwave-enhanced and totally compatible with SDS-PAGE sample buffer and IEF rehydration buffer. The 390

Journal of Proteome Research • Vol. 6, No. 1, 2007

Acknowledgment. We thank members of the Laboratory of Molecular Biology at Tsinghua University for many insightful discussions. We would like to acknowledge the State KeyBasicResearchandDevelopmentPlanofChina(2004CB117303 and 2006CB101706), the Specialized Research Fund for the Doctoral Program of Higher Education (20050003066), and the National Natural Science Foundation of China (30370847) for their support of this work. References (1) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-54. (2) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.; Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N. M.; Olson, B. J.; Klenk, D. C. Measurement of protein using bicinchoninic acid. Anal. Biochem. 1985, 150 (1), 76-85. (3) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193 (1), 265-75. (4) Sheffield, J. B.; Graff, D.; Li, H. P. A solid-phase method for the quantitation of protein in the presence of sodium dodecyl sulfate and other interfering substances. Anal. Biochem. 1987, 166 (1), 49-54.

technical notes (5) Lim, M. J.; Patton, W. F.; Shojaee, N.; Shepro, D. Solid-phase metal chelate assay for quantifying total protein: resistance to chemical interference. Biotechniques 1996, 21 (5), 888-92, 894, 896-7. (6) Kuno, H.; Kihara, H. K. Simple microassay of protein with membrane filter. Nature 1967, 215 (104), 974-5. (7) Morcol, T.; Subramanian, A. A red-dot-blot protein assay technique in the low nanogram range. Anal. Biochem. 1999, 270 (1), 75-82. (8) Agnew, B. J.; Murray, D.; Patton, W. F. A rapid solid-phase fluorescence-based protein assay for quantitation of protein electrophoresis samples containing detergents, chaotropes, dyes, and reducing agents. Electrophoresis 2004, 25 (15), 2478-85. (9) Nishibu, T.; Hirayasu, K.; Tanaka, T.; Takeda, Y.; Kobayashi, Y. Quantitative filtration-blotting of protein in the presence of sodium dodecyl sulfate and its use for protein assays. Anal. Biochem. 2003, 319 (1), 88-95. (10) Hunter, J. B.; Hunter, S. M. Quantification of proteins in the low nanogram range by staining with the colloidal gold stain AuroDye. Anal. Biochem. 1987, 164 (2), 430-3. (11) Shojaee, N.; Patton, W. F.; Lim, M. J.; Shepro, D. Pyrogallol redmolybdate: a reversible, metal chelate stain for detection of proteins immobilized on membrane supports. Electrophoresis 1996, 17 (4), 687-93. (12) Kihara, H. K.; Kuno, H. Microassay of protein with nitrocellulose membrane filters. Anal. Biochem. 1968, 24 (1), 96-105. (13) Lim, M. J.; Patton, W. F.; Lopez, M. F.; Spofford, K. H.; Shojaee, N.; Shepro, D. A luminescent europium complex for the sensitive detection of proteins and nucleic acids immobilized on membrane supports. Anal. Biochem. 1997, 245 (2), 184-95. (14) Minamide, L. S.; Bamburg, J. R. A filter paper dye-binding assay for quantitative determination of protein without interference from reducing agents or detergents. Anal. Biochem. 1990, 190 (1), 66-70.

Wu et al. (15) Mitra, P.; Pal, A. K.; Basu, D.; Hati, R. N. A staining procedure using Coomassie brilliant blue G-250 in phosphoric acid for detection of protein bands with high resolution in polyacrylamide gel and nitrocellulose membrane. Anal. Biochem. 1994, 223 (2), 327-9. (16) Hancock, K.; Tsang, V. C. India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 1983, 133 (1), 157-62. (17) Kovarik, A.; Hlubinova, K.; Vrbenska, A.; Prachar, J. An improved colloidal silver staining method of protein blots on nitrocellulose membranes. Folia Biol. (Prague) 1987, 33 (4), 253-7. (18) Moeremans, M.; De Raeymaeker, M.; Daneels, G.; De Mey, J. FerriDye: Colloidal iron binding followed by Perls’ reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes. Anal. Biochem. 1986, 153 (1), 18-22. (19) Hua, L.; Low, T. Y.; Sze, S. K. Microwave-assisted specific chemical digestion for rapid protein identification. Proteomics 2006, 6 (2), 586-91. (20) Sun, W.; Gao, S.; Wang, L.; Chen, Y.; Wu, S.; Wang, X.; Zheng, D.; Gao, Y. Microwave-assisted protein preparation and enzymatic digestion in proteomics. Mol. Cell. Proteomics 2006, 5 (4), 76976. (21) Zhong, H.; Marcus, S. L.; Li, L. Microwave-assisted acid hydrolysis of proteins combined with liquid chromatography MALDI MS/ MS for protein identification. J. Am. Soc. Mass Spectrom. 2005, 16 (4), 471-81. (22) Zhong, H.; Zhang, Y.; Wen, Z.; Li, L. Protein sequencing by mass analysis of polypeptide ladders after controlled protein hydrolysis. Nat. Biotechnol. 2004, 22 (10), 1291-6.

PR0602848

Journal of Proteome Research • Vol. 6, No. 1, 2007 391