Coomassie Brilliant Blue Removal/Disposal from Gel Destain and ...

Chapter 55 Coomassie Brilliant Blue Removal/Disposal from Gel Destain and Used Gel Stain in an Environment-Friendly Manner Biji T. Kurien and Yaser Dorri Abstract Toxic reagents are employed to destain Coomassie Brilliant Blue (CBB) stained gels. We tested the efficacy of various paper adsorbents in adsorbing CBB released from gels during destaining. Kimwipes were the most efficient, followed by Teri towels, multifold towels, and Whatman (numbers 1 and 3) filter papers. Three Kimwipes added during destaining of a CBB-stained mini-gel helped adsorb the released dye. Thus, stain removal with Kimwipes helps reduce destain use and organic waste accumulation, enables recycling of nonradioactive destaining solution, and is 7.5-fold cheaper than an available method for CBB disposal. Next, we used Kimwipes to deplete the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. Seventy-five Kimwipes successfully helped remove the dye from a 0.05% CBB staining solution in 5 to 10 min. The blue-colored Kimwipes did not release the stain even when squeezed dry after incubation in various salts, water, or acid solutions for five weeks. The CBB removed thus can be simply disposed as solid waste and will not leach out from solid landfills. Kimwipes, thus, enables CBB disposal in an environmentally friendly manner and allows recycling of destaining solution. Key words: SDS-PAGE, 2-Dimensional gel electrophoresis, Coomassie Brilliant Blue, Gel destaining, Kimwipes, Environment-friendly destaining

1. Introduction Laboratories involved in proteomics commonly employ sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (1) and two-dimensional gel electrophoresis (2-DE) (2) to analyze proteins. Proteins separated by SDS-PAGE/2-DE are usually visualized by Coomassie Brilliant Blue (CBB) stain due to its simplicity and reliability (3). Methanol (4, 5) or isopropanol (6), along with acetic acid fixative, is commonly used to destain CBB-stained gels. As the gel loses its excess stain, the destaining solution becomes

Biji T. Kurien and R. Hal Scofield (eds.), Protein Electrophoresis: Methods and Protocols, Methods in Molecular Biology, vol. 869, DOI 10.1007/978-1-61779-821-4_55, © Springer Science+Business Media, LLC 2012

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colored and normally needs to be replaced with fresh destaining solution multiple times for efficient destaining. CBB has been reported to be toxic (7) and requires expensive special treatment for disposal. Similarly, acetic acid and methanol are also toxic and cannot be disposed into the sink. The fact that these toxic reagents can ultimately get into drinking water supplies poses an environmental hazard. Disposal of small quantities of 10% acetic acid is allowed, following neutralization to pH 7 (http://www.scholarchemistry. com/msds/Acetic_Acid_10pct.pdf). Special waste disposal procedures are recommended for even buffers with only 10% methanol, which are thought to be hazardous (8). Procedures to dispose of these reagents are expensive and time-consuming. We have used a paper product (9) to safely eliminate CBB from the destain solution in a simple fashion that allows acetic acid and methanol to be recycled. This work resulted from the observation that the inclusion of two multifold towels in the destaining solution helped to adsorb most of the stain. Subsequently, we compared the efficiency of stain removal of two multifold towels with equivalent weights of Kimwipes, Whatman numbers 1 and 3 filter papers, and Teri towels. Kimwipes were found to be the most efficient, followed by Teri towels, multifold towels, Whatman number 1, and Whatman number 3 in decreasing order. Charcoal cartridges have been employed for removing anionic detergents and CBB (10). In addition, activated charcoal has been employed to adsorb stain. But, this procedure (http://www.p2000. umich.edu/chemical_waste/cw4.htm) seems cumbersome compared to our procedure. In addition, there is the added disadvantage of particulate release when using activated charcoal (11). Cozap, a spongelike material, is packed in a Mylar envelope and sold commercially for CBB stain removal by adsorption. It (http:// www.bioexpress.com/index.html? wscdet_show = 000000000100 114000114050) costs about $3.12 per piece, with each piece reportedly capable of destaining 10 mini-gels at the cost of about 30 cents/gel. (http://www.the-scientist.com/article/display/17674/). However, it costs slightly less than 4 cents/gel to destain gels using Kimwipes. Thus, destaining with Kimwipes is 7.5-fold cheaper compared to Cozap, as well as being easier and cleaner. Then, we also investigated the ability of Kimwipes to remove the dye from a used CBB staining solution. Laboratories routinely collect used CBB stain as organic waste, following gel staining, for proper disposal by the Institutional Safety Office. A licensed professional waste disposal service is to be contacted, according to regulations, to dispose such material. Disposal regulations stipulate (in the United States and Europe) that this combustible material may be burned in a chemical incinerator equipped with an afterburner and a scrubber following all federal, state, and local environmental rules (http://www.teknova.com/v/vspfiles/files/

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MSDS/C1040.pdf). Such methods are time-consuming and expensive. Kimwipes were found to efficiently remove nearly all of the dye from the used stain in 5–10 min in a manner similar to the removal of CBB from the destaining solution. The CBB removed thus remains tightly bound to the Kimwipes and does not leach out in 1 M salts, water, or acidic solutions even after lengthy incubation. This procedure allows the disposal of CBB as solid waste and thus helps to keep the environment safer, with little or no chance for toxic reagents and chemicals to contaminate drinking water reservoirs. Removal of CBB from nonradioactive destaining solution in this fashion permits it to be recycled for further use.

2. Materials Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 M Ω cm at 25°C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials. We do not add sodium azide to reagents. 1. 10% SDS-PAGE gels (1-mm thickness). 2. CBB stain stock: 0.25% CBB in water (see Note 1). 3. CBB stain (0.05%): Dilute 0.25% CBB stain with water to obtain 0.05% stain. 4. CBB stain: 0.25% CBB in 25% methanol/10% acetic acid (see Note 2). 5. Destaining solution: 25% methanol/10% acetic acid. 6. Prestained molecular weight protein standards. 7. HeLa cell extract. 8. SDS-PAGE running buffer: 0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3 (see Note 3). 9. SDS lysis buffer (5×): 0.3 M Tris–HCl (pH 6.8), 10% SDS, 25% β-mercaptoethanol, 0.1% bromophenol blue, 45% glycerol. Leave one aliquot at 4°C for current use and store remaining aliquots at −20°C (see Note 4). 10. Bromophenol blue (BPB) solution: Dissolve 0.1 g BPB in 100 mL water. 11. Phosphate buffered saline (PBS), pH 7.4. 12. Branson Sonifier Cell Disruptor 185 (VWR Scientific Co., Boston, MA, USA). 13. FB300 power supply (Fisher Scientific, Houston, TX, USA). 14. Kimwipes (Kimberly–Clarke, Roswell, GA, USA).

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15. Tradition multifold towels (Kimberly–Clarke). 16. Whatman filter paper (numbers 1 and 3). 17. Teri towels (see Note 5). 18. Fisher Scientific balance (XE series, model XL100).

3. Methods All procedures are carried out at room temperature unless otherwise specified. 3.1. Determining CBB Adsorption by Different Paper Adsorbents

1. Take 250 mL of water in each of five identical plastic containers. 2. Add 5 mL of 0.25% CBB solution (dissolved in water) to each container and mix well. Save an aliquot (1 mL) from each container to measure zero time optical density of the diluted CBB solution at 560 nm 3. Weigh two Tradition multifold towels in a Scientific balance. Weigh equal weights (see Note 6) of Kimwipes (~10.5 counts), Teri towels (approximately half), and Whatman filter papers (numbers 1 and 3). 4. Add each paper product to one of the five containers and incubate with gentle shaking at room temperature for 90 min (see Note 7). 5. Aliquots (1 mL) were taken from each container at periodic intervals and finally at the end of 90 min (see Table 1). 6. Read optical density of the saved samples at 560 nm using a Spectronic Genesys 5 Spectrophotometer (see Table 1).

3.2. Destaining CBB-Stained SDSPAGE Mini-Gel with Kimwipes

1. Electrophorese aliquots of HeLa cell extract (see Note 8) and bovine serum albumin on 10% (1 mm thick) SDS-PAGE gel. 2. Stain with CBB stain in a plastic container (see Note 9). Remove the used CBB stain into a container employed for collecting used CBB. 3. Rinse the CBB off the gel with few mL of ultrapure water prior to adding destaining solution (see Note 10). 4. Insert the CBB-stained gel between the folds of three Kimwipes (see Fig. 1 and Note 11) and place the gel-Kimwipe sandwich into another plastic container. 5. Add 200 mL of destaining solution into the container containing the gel-Kimwipe sandwich. Close the container tightly and destain the gel at room temperature with gentle shaking (see Note 12). The Kimwipes will readily adsorb all the stain released by the gel (see Fig. 1).

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Table 1 CBB adsorption from a solution containing excess CBB by various paper products. The CBB was dissolved in ultrapure water only. Since there was no gel involved, we did not use either acetic acid or methanol. The residual amount of stain remaining after incubation with the different paper products at the different timings was measured at 560 nm. The optical density of the stain prior to the addition of paper adsorbents is shown at zero time Optical density at 560 nma Product

0 min

10 min

30 min

90 min

3 M Whatman

1.75

1.62

1.61

1.6

1 M Whatman

1.75

1.55

1.55

1.53

Multifold towels

1.77

1.26

1.06

0.84

Teri towels

1.76

0.6

0.39

0.25

Kimwipes

1.67

0.36

0.114

0.04

Reproduced from (9) with permission from Elsevier Limited, Oxford, OX5 1 GB, UK a Values are means of three determinations

3.3. Effect of Prolonged Incubation of CBB-Stained Kimwipes in Water and Salt Solutions

1. Prepare CBB-stained Kimwipes by incubating fresh Kimwipes in 0.05% CBB (see Note 1). 2. Rinse the intensely blue-colored Kimwipes in water to remove any nonadherent color. 3. Incubate CBB-stained Kimwipes in water for 5 weeks at room temperature. 4. Incubate CBB-stained Kimwipes also in 1 M sodium bicarbonate, 1 M sodium chloride, or 1 M sodium phosphate (dibasic anhydrous) for 5 weeks at room temperature. 5. Wring the Kimwipes dry at the end of experimental period (see Note 13).

3.4. CBB Removal from an Experimental Gel-Staining Solution with Kimwipes

1. Pour 250 mL of 0.05% CBB stain (dissolved in water) into a 1-L beaker. 2. Add 15 Kimwipes into the solution in the beaker, one at a time in quick succession. 3. Swirl the Kimwipes in the solution with a glass rod for a minute or less. 4. Remove the colored Kimwipes from the beaker and squeeze dry. Repeat this procedure four more times, each time with 15 fresh Kimwipes (see Notes 13–15 and Fig. 2).

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Fig. 1. Destaining of CBB-stained gel using Kimwipes and destain solution. (a) The CBB-stained gel is positioned over one-half of Kimwipes (3 sheets) in destaining solution. (b) The gel is covered completely with the other half of Kimwipes. (c) Stained gel inserted between Kimwipes prior to destaining. (d) Kimwipes with CBB adsorbed from the gel. Reproduced from (9) with permission from Elsevier Limited, Oxford, OX5 1 GB, UK.

Fig. 2. Rapid CBB removal from gel stain using Kimwipes. Kimwipes can be employed to deplete CBB from used gel stain earmarked as organic waste for proper disposal by the Institutional Safety Office. Kimwipes (15 numbers used each time) were dipped into a 1-L glass beaker containing 250 mL of 0.05% CBB dissolved in water. Each time, they were removed immediately with a glass rod and wrung dry (a–e). The majority of the stain was adsorbed by the first set of Kimwipes (a). Kimwipes shown in B-E can be saved for adsorbing more CBB. Reproduced from (9) with permission from Elsevier Limited, Oxford, OX5 1 GB, UK.

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4. Notes 1. Prepare CBB without methanol or acetic acid since this solution is to be used to test the adsorption capacity of various paper products such as Kimwipes, Teri towels, multifold paper towels, and 1- and 3-M Whatman filter papers. Filter (0.22-μm cellulose acetate membrane filter) the reagent to remove any insoluble CBB particles. This solution is not to be used for staining gels. 2. Weigh the required amount of the dye and dissolve it in methanol first. Filter the dissolved CBB through a circular Whatman 1-M filter paper folded into a cone and fitted inside a glass funnel. For a quicker way to filter the CBB solution, layer a circular 1-M Whatman filter paper (125-mm-diameter circles) inside a Buchner funnel (Coors, USA; 135-mm inner diameter). The filter paper should cover all the holes in the funnel and lay flat inside the funnel. Attach the Buchner funnel to a side-arm fitted conical flask that is attached to the house vacuum through the side-arm. Pour the CBB solution into the Buchner funnel and apply vacuum. If filter paper gets clogged, replace with a fresh one and filter. Collect the filtered CBB. Adjust volume with water, methanol, and acetic acid to obtain a 0.05% Coomassie in 25% methanol/10% acetic acid. Make sure to add acetic acid to water and not vice-versa. 3. Simple method of preparing SDS-PAGE running buffer: Prepare 10× native buffer (0.25 M Tris, 1.92 M glycine). Weigh 30.3 g Tris and 144 g glycine, mix and make it to 1 L with water. Do not adjust pH. Dilute 100 mL of 10× native buffer to 990 mL with water in a graduated cylinder and add 10 mL of 10% SDS. Care should be taken to add SDS solution last since it makes bubbles. Mix the contents of the graduated cylinder using a magnetic stirrer or manually by hand (cap the graduated cylinder with Parafilm and mix by inverting several times). 4. SDS precipitates at 4°C. Therefore, the lysis buffer needs to be warmed prior to use. 5. Kimwipes, Tradition multifold paper towels, Whatman paper, and Teri towels can be purchased from Fisher Scientific Co., Dallas, TX, USA. 6. Weigh enough Kimwipes to equal the weight of two Tradition multifold towels. Weigh the other paper products similarly. 7. Kimwipes (about 10) was found to be the most efficient, adsorbing almost the entire stain in 90 min. It took up more than three-fourths of the stain in about 10 min (see Table 1). An increase in surface area (e.g., that provided by ~10 Kimwipes) combined with the good adsorption property of

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Kimwipes appears to be the reason for the efficient adsorption of the CBB stain. The order of adsorbing efficiency was Kimwipes > Teri towels > multifold towels > Whatman No. 1 > Whatman No. 3. Whatman filters did not take up any significant stain and also left behind some turbidity (particularly Whatman No. 1 filter paper) resulting from apparent onset of disintegration of the filter paper with increase in incubation time. 8. Preparation of HeLa cell extract: Harvest freshly cultured HeLa cells by centrifuging at 800 × g and wash twice with PBS. Lyse cells by sonication in PBS buffer using a Branson sonicator (setting 4) and centrifuge at 10, 000 × g for 10 min. Use an aliquot of the supernatant for SDS-PAGE. Sonicators generate high-frequency sound waves in the 20,000 Hz range, outside our normal range of hearing. These sound waves can cause hearing damage, and therefore, laboratory personnel should wear sound mufflers when sonication is in process (http://www.labmanager.com/?articles.view/articleNo/1103/ article/Sonicator-Safety). Cool microcentrifuge tube, containing HeLa cells suspended in PBS, on ice first. Clean sonicator probe with ethanol first and then with distilled water. Wipe the probe dry with Kimwipe. Sonicate for 10 s and let cool on ice. Repeat this step three more times. Make sure that the probe does not touch the bottom of the tube when sonication is in progress to avoid the probe from puncturing a hole in the tube. 9. The gel can be heated at 65°C in a water bath for 20 min. Heating has been shown to accelerate protein staining and increasing the sensitivity of staining (12). If controlled heating temperatures are not essential, the plastic container with the CBB stain and the gel can be heated using a microwave. However, care needs to be taken to not overheat the container in the microwave. Also, the container needs to be covered tightly to prevent evaporation of acetic acid and to prevent acetic acid fumes from contaminating the laboratory. 10. Do not discard the wash into the sink. Save the rinse in a bottle for proper disposal (see Subsection 3.4). 11. It is not necessary to place the gel within the folds of the Kimwipes to enable adsorption of the dye. Kimwipes can be moistened in the destaining solution and placed as a crumpled ball in one corner of the container for CBB removal. However, inserting the gel between the folds of the Kimwipes enables the adsorption of the CBB as it is released from the gel. 12. The use of heat has been shown to accelerate the destaining process. It takes approximately 15–30 min to destain with the application of heat (12).

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13. The Kimwipes will not leach any of the bound Kimwipe into water or any of the salts used. The CBB remains tightly bound to the Kimwipes even after lengthy incubation in salts or water, at various pH values used in these experiments (pH 2.4 with CBB destaining solution, pH 7.95 with sodium bicarbonate, and pH 8.8 with sodium phosphate [dibasic anhydrous]). Thus, CBB will not leach out from the Kimwipes dumped at solid waste landfills even under conditions of high salt concentration or highly acidic conditions. 14. Thus, the CBB-stained Kimwipes can be discarded as solid waste and will not release the bound CBB during rains or in situations involving high salts. 15. Almost all the dye from a 0.05% CBB staining solution could be removed with 75 Kimwipes (see Fig. 2) in just 5–10 min. Interestingly, the Kimwipes adsorb the stain quickly when there is excess CBB dye present, as seen with the 0.05% staining solution. Once the dye level decreases, the stain adsorbed by the Kimwipes quickly is reduced. This can be seen in Fig. 2 where the intensity of color decreases from Fig. 2a–e. Therefore, when using dilute CBB solutions, as obtained with gel destaining, it is better to leave the Kimwipes in the container for a longer time (the duration of gel destaining).

Acknowledgments This work was supported in part by an Oklahoma Center for the Advancement of Science and Technology grant to B.T.K. We thank Hal Scofield (Oklahoma Medical Research Foundation [OMRF]) for his support of this work. B.T.K. first started using multifold paper towels during the early 1990s to remove CBB from gel destain in Hiroyuki Matsumoto’s laboratory. We also thank Justin Simmons, safety officer at OMRF, for his input regarding proper disposal of CBB in the institutional setting. References 1. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 2. Dorri Y, Kurien BT, Scofield RH (2009) A simpler and faster version of two-dimensional gel electrophoresis using vertical, mini SDSPAGE apparatus. Iran J Chem Chem Eng 28:51–56

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9. Dorri Y, Kurien BT (2010) Environmentally safe removal/disposal of Coomassie Brilliant Blue from gel destain and used gel stain. Anal Biochem 404:193–196 10. Duhamel RC, Meezan E, Brendel K (1981) A charcoal cartridge for the removal of anionic detergent and electrophoresis stains. J Biochem Biophys Methods 4:73–80 11. Chandy T, Sharma CP (1998) Activated charcoal microcapsules and their applications. J Biomater Appl 13:128–157 12. Kurien BT, Scofield RH (1998) Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels. Indian J Biochem Biophys 35:385–389