Article pubs.acs.org/jchemeduc
Electrophoresis Gel Quantification with a Flatbed Scanner and Versatile Lighting from a Screen Scavenged from a Liquid Crystal Display (LCD) Monitor Brendan Yeung,§ Tuck Wah Ng,*,§ Han Yen Tan,‡ and Oi Wah Liew† †
Cardiovascular Biomarkers Laboratory, Cardiovascular Research Institute, 30 Medical Drive, Singapore 117609 Department of Ophthalmology, SUNY Upstate Medical University, Syracuse, New York 13210, United States § Laboratory for Optics, Acoustics and Mechanics, Monash University, VIC 3800, Australia ‡
ABSTRACT: The use of different types of stains in the quantification of proteins separated on gels using electrophoresis offers the capability of deriving good outcomes in terms of linear dynamic range, sensitivity, and compatibility with specific proteins. An inexpensive, simple, and versatile lighting system based on liquid crystal display backlighting is described. It is used with a flatbed scanner technique previously reported. The results from experimentation and analysis were benchmarked against a commercial densitometer to verify the performance of this analysis system. The availability of this device is expected to be useful in laboratories in schools and in resource-limited venues.
KEYWORDS: Upper-Division Undergraduate, Analytical Chemistry, Biochemistry, Electrophoresis, Laboratory Equipment/Apparatus, Proteins/Peptides, Quantitative Analysis
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he state of the art of densitometry has been reviewed1 in which inexpensive flatbed scanners offer some advantages in application.2−6 In the case of their application for densitometry of stained polyacrylamide electrophoresis gels, the investigation of the relevant factors and their attendant proper use has yielded analysis outcomes that can be as good as, if not better than, those of commercial densitometers.6−9 One of these factors is related to the use of appropriate lighting. As the information contained in the stained gels appears as a two-dimensional format, it is imperative to have the light source operating not only at the desired wavelength but also with a spatially uniform distribution for consistent measurements. The use of adapted backlighting from liquid crystal display (LCD) monitors has been recently shown to be superior to a twodimensional array of light-emitting diodes (LEDs) as it provides a compact architecture, obviates the soldering of multiple LEDs and production of a printed circuit board to house the LEDs, and limits light loss due to attenuation with diffusers.9 A study of how the recording and illumination conditions can affect measurement sensitivity was also conducted.7 An instrumental design is described in which different wavelength light sources can be interchanged. This flatbed scanner densitometry can be used beyond the analysis of Coomassie-stained gels. LCD backlighting predominantly applies linear cold cathode fluorescent lamps mounted on two edges of the display. Light from these lamps is collected from the two edges into a plastic © 2012 American Chemical Society and Division of Chemical Education, Inc.
lightpipe. The plastic lightpipe typically has a series of printed or implanted reflectors that helps to scatter and channel the light so that it emanates from both surfaces. The lightpipe is often backed with a mirror at one surface to allow almost all light to appear from the opposite end. Typically an electronically addressable liquid crystal unit and polarizer are placed in that order above the lightpipe surface to permit texts and pictures to be displayed. The removal of these two elements results in a compact device that emits a spatially uniform distribution of light. Generally, most LCD monitors can be dismantled by removing the screws. Once they are dismantled, there will be reflector sheets and the lightpipe. Both the lightpipe and reflector sheets are important to ensure that illumination remains uniform throughout the lightpipe. The circuitry that is used to control the liquid crystals can be discarded. The lighting tubes need to be powered with 12 V dc and draw about 700 mA. They are normally supplied as a pair and come with prewired rocker switch and a small sealed inverter that is prewired with a 280 mm long cable to the tube and a 540 mm power cord terminated to a standard computer hard drive power plug socket. One can power the lighting tubes from either the computer or an external power supply. Published: January 27, 2012 513
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HARDWARE ASSEMBLY
accomplished using garage tools such as handsaws and hand drills. The frame design allowed position adjustment of the horizontal members prior to assembly for optimal contact with the lightpipe. The construction makes use of epoxy adhesive to ease the assembly process, while maintaining structural rigidity. Wing-nuts on the slider mechanism aided in convenient adjustment of the lighting tubes, while positioning with washers as spacers permitted optimal lighting height to be achieved to maximize light uniformity within the lightpipe. Another feature is the addition of wall plugs in the frame to prevent wood grain damage from repetitive tightening of screws. The completed system minimizes optical distortion by having the electrophoresis gel sample sitting closely between the lightpipe and the adapted flatbed scanner, reducing the probability of dust particle and undesired light interference.
An illustration of the component breakdown of the lighting unit developed is shown in Figure 1. Three major features are
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SAMPLE PREPARATION The protein sample used for the experiment was recombinant His6-tagged fusion protein (molecular weight: 29.4 kDa) that was purified by immobilized metal affinity chromatography to greater than 95% purity and quantified by the BCA assay (Pierce, Rockford IL, USA) according to manufacturer’s instructions.10 A series of dilutions of the recombinant His6tagged fusion protein sample were prepared, and the quantity of proteins loaded per well ranged from 50 to 800 ng. These were resolved on a 16% discontinuous SDS−polyacrylamide slab gel prepared using Laemmli’s11 method. Vertical electrophoresis was carried out using a Mini-Protean Electrophoresis Cell (Bio-Rad, Hercules, CA, USA) using the manufacturer’s instructions12 on the two similar polyacrylamide gels. The first polyacrylamide gel was stained overnight with colloidal Coomassie G-250 (Sigma-Aldrich) and subjected to a water wash enhancement step where the stain was replaced with several changes of ultrapure water until a clear background was achieved.13 Apart from Coomassie, there are other varieties of stains available for protein visualization, quantification, and characterization.14 One of the popular stains is the SYPRO Ruby fluorescent stain, which is known for its broad linear dynamic ranges, high sensitivity, and compatibility with downstream protein identification or characterization technologies.14,15 In addition, the advantages of SYPRO Ruby protein gel stain over silver stain have been demonstrated as well,16 leading to its application in gel-based proteomics.17 The second polyacrylamide gel was stained overnight using SYPRO Ruby (Bio-Rad) and rinsed with 10% ethanol and 7% acetic acid to decrease the background fluorescence of the gel. The gel was then washed in water before imaging.
Figure 1. The components (as depicted in the legend) of the lighting unit developed. The advantageous features are its low cost, light weight, brightness control capability, and ability for wavelength selection (via interchanging the fluorescent lamp assembly).
highlighted. First, the four pieces making up the frames are of lightweight material (e.g., chip wood) and can be joined together using adhesive, staples, or small nails. Second, the pair of slotted guides that attach to each fluorescent lamp permit adjustable movement to control brightness. In addition, the lamp can be interchanged with another lamp having a different output color. Third, the soft spacers serve to provide positive attachment between screw and lightpipe without damaging the latter. An image of the lighting unit working in conjunction with an adapted flatbed scanner is shown in Figure 2. It should
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HAZARDS
The manufacture of the lighting frame involves the use of drilling and cutting tools. This requires personal protective equipment (PPE) in the form of safety glasses, workshop coat, and shoes to be worn to minimize accidental physical harm. Some training in use of garage tools is recommended. Any harm to the user is minimal in assembling the system and is further reduced during usage. As the frame was joined using an epoxy adhesive, it is advisible for the work area to be well ventilated as excessive inhaling of the fumes may cause respiratory irritation and dizziness. Skin contact with the substance can cause irritation, and in some cases exposure to the eyes may cause blindness. It is therefore highly
Figure 2. The lighting unit working in conjunction with an adapted flatbed scanner. The lighting unit may be placed below the scanner or vice versa.
be noted that the lighting unit can be placed below the scanner or vice versa. The underlying objective in the creation of the lighting unit is to ensure simplicity and to minimize costs. The lightpipe was salvaged from a discarded LCD monitor, and all the materials used were acquired from a neighborhood hardware store. Manufacture of the components of the system can be 514
dx.doi.org/10.1021/ed101173k | J. Chem. Educ. 2012, 89, 513−516
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and a commercial densitometer (AlphaImager). Recordings for each gel were conducted five times for both systems to evaluate sensitivity and repeatability. In the process, the precautions as outlined in a previous work were adhered to.7 The images were processed using the area density function found in the software ImageJ.18 A red cold cathode fluorescent lamp (Jaycar Electronics, Australia) was used for Coomassie stained gel recording with the flatbed scanner, in the vein of matching the wavelengths according to maximal absorbance.6−9 In the case of the SYPRO Ruby stained gel recording, the light source used (with the flatbed scanner) was selected based on matching the excitation wavelength of the dye with the illumination wavelength of available fluorescent light sources.
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Figure 3. Measured illumination spectrum from blue and UV fluorescent lamps, in which the former was found to be more amenable for exciting the SYPRO Ruby fluorescent stain.
RESULTS AND DISCUSSION The output spectra from a spectrometer (Ocean Optics, USB4000) in which a UV and blue fluorescent lamp were used are shown in Figure 3. It can be seen that the blue fluorescent light is more amenable for exciting the SYPRO Ruby stained gel than the UV light. The average area density readings obtained from the Coomassie-stained gels using the lighting unit and flatbed scanner system, as well as the AlphaImager, are shown in Figure 4A. The former is seen to be marginally more sensitive, as denoted by the fitted slope values of 18.47 versus 16.58. Nevertheless, the linearity trends of both were equally high above 0.99. The situation is reversed with the SYPRO Ruby-stained gel (Figure 4B) wherein the AlphaImager sensitivity was marked higher with a slope value of 19.96 versus 11.60. This is due to the relatively broad spectrum of the blue fluorescent light (Figure 3) used with the flatbed scanner. The linearity values, however, were both equally high and above
recommended that gloves and safety glasses be worn during the assembly phase. The hazards in the operation phase are minimal and are no greater than the hazards of using a typical household electronic appliance. It is generally not a good practice to handle electrophoresis gel samples without wearing gloves. When using UV lamps as a light source, it is necessary to wear some protective eyewear. When dismantling an LCD monitor, the power must always be switched off.
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GEL SCANNING The basis of how the lighting is used in transmission mode for flatbed scanning of gels has been described previously.9 Both gels were imaged using the lighting system with flatbed scanner
Figure 4. The average area density (from five measurements) against protein mass determined from the (A) Coomassie and (B) SYPRO Ruby stained gels using the lighting unit and flatbed scanner system, and AlphaImager. The corresponding calculations of standard deviation/average (from the five measurements) are presented for the (C) Coomassie and (D) SYPRO Ruby stained gels, respectively. 515
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(7) Tan, H. Y.; Ng, T. W.; Liew, O. W. Appl. Opt. 2010, 49, 1623− 1629. (8) Tan, H. Y.; Ng, T. W. Opt. Commun. 2008, 281, 3013−3017. (9) Tan, H. Y.; Ng, T. W.; Liew, O. W. Electrophoresis 2009, 30, 987− 990. (10) Instructions for the Pierce BCA Protein Assay Kit. http://www. piercenet.com/instructions/2161296.pdf (accessed Dec 2011). (11) Laemmli, U. K. Nature (London) 1970, 227, 680−685. (12) Instruction for vertical electrophoresis. www.bio-rad.com/ webroot/web/pdf/lsr/literature/10007296.PDF (accessed Dec 2011). (13) Instructions for the GelCode Blue Stain Reagent. http://www. piercenet.com/instructions/2160714.pdf (accessed Dec 2011). (14) Chevalier, F.; Rofidal, V.; Rossignol, M. Methods Mol. Biol. 2007, 355, 145−156. (15) Harris, L. R.; Churchward, M. A.; Butt, R. H.; Coorssen, J. R. J. Proteome Res. 2007, 6, 1418−1425. (16) Lopez, M. F.; Berggren, K.; Chernokalskaya, E.; Lazarev, A.; Robinson, M.; Patton, W. F. Electrophoresis 2000, 21, 3673−3683. (17) Yang, Y.; Wang, J.; Bu, D.; Zhang, L.; Li, S.; Zhou, L.; Wei, H. A fluorescence-based Coomassie Blue protocol for two-dimensional gelbased proteomics. Biotechnol. Lett. 2011, 33, 119−121. (18) Software ImageJ. http://rsb.info.nih.gov/ij/download.html (accessed Dec 2011). (19) Patton, W. F. J. Chromatogr., B 2002, 771, 3−31. (20) Nishihara, J. C.; Champion, K. M. Electrophoresis 2002, 23, 2203−2215. (21) Chiangjong, W.; Thongboonkerd, V. J. Chromatogr., B 2009, 877, 1433−1439. (22) Chevalier, F.; Rofidal, V.; Vanova, P.; Bergoin, A.; Rossignol, M. Phytochemistry 2004, 65, 1499−1506. (23) Luo, S.; Wehr, N. B.; Levine, R. L. Anal. Biochem. 2006, 350, 233−238. (24) Anderson, G.; Thompson, J. E.; Shurrush, K. J. Chem. Educ. 2006, 83, 1677−1680.
0.98. These results indicate that the lighting unit and flatbed scanner system compares favorably in performance with the commercial AlphaImager. The corresponding calculations of standard deviation/average (from the five measurements) are presented for the Coomassie (Figure 4C) and SYPRO Ruby (Figure 4D) stained gels, respectively. In both cases, a reducing trend (indicative of improved repeatability) can be seen with increase in protein mass. This result is generally expected due to the known limits of detection for colloidal Coomassie Blue ranging between 30 and 100 ng depending on the protein stained.19 The only data point that did not conform to this was the SYPRO Ruby flatbed scanning at 50 ng. Possibly the scanner sensor was operating just below the threshold of detectability at this low fluorescence emission level. This has the effect of causing all readings to falsely possess lower levels of deviation. Furthermore, very low quantities of proteins may be detectable but do not fall within the dynamic linearity range because staining is not linear near the lower limit of detection.20 If one adopts an SD value of 0.1 as a reasonable benchmark, it is interesting to note that measurements with Coomassie appear to have lower repeatability when the protein concentration was below 100 ng. This highlights the effectiveness and greater sensitivity of using fluorescent stains compared with Coomassie stains when dealing with such situations21,22 although the performance of Coomassie can be substantially enhanced when IR fluorescence imaging is used instead of conventional densitometry.15,23 An inexpensive capillary electrophoresis system previously reported24 might benefit from this system.
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CONCLUSIONS A versatile, simple, and inexpensive lighting system that can be used with flatbed scanners is described. This system was able to extend the usage of flatbed scanners to the densitometry of fluorescent stains. Good results were achieved when benchmarked with a commercial densitometer. This system will be particularly useful in biochemical laboratories that do not have the monetary resources to acquire expensive instrumentation such as in schools and resource-limited countries.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
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ACKNOWLEDGMENTS T.W.N. appreciates the funding support provided by the Monash NSMF scheme. Preliminary discussions with Adrian Neild are noted.
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REFERENCES
(1) Berth, M.; Moser, F. M.; Kolbe, M.; Bernhardt, J. Appl. Microbiol. Biotechnol. 2008, 72, 1223−1243. (2) Pembleton, K. G.; Volenec, J. J.; Rawnsley, R. P.; Donaghy, D. J. Crop Sci. 2010, 72, 989−999. (3) Jäger, G.; Wu, Z.; Garschhammer, K.; Engel, P.; Klement, T.; Rinaldi, R.; Spiess, A. C.; Büchs, J. Biotechnol. Biofuels 2010, 3, 18. (4) Gassmann, M.; Grenacher, B.; Rohde, B.; Vogel, J. Electrophoresis 2009, 30, 1845−1855. (5) Nishizuka, S.; Washburn, N. R.; Munson, P. J. BioTechniques 2006, 40, 442−447. (6) Tan, H. Y.; Ng, T. W.; Liew, O. W. BioTechniques 2007, 42, 474− 478. 516
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