Anal. Chem. 2007, 79, 9478-9483
Laser-Induced Fluorescence Detector for Capillary-Based Isoelectric Immunoblot Assay James E. Knittle,* David Roach, Peter B. Vander Horn, and Karl O. Voss
Cell Biosciences, Inc., 1050 Page Mill Road, Palo Alto, California 94304
We describe a whole-capillary, multicolor laser-induced fluorescence scanner for microfluidic protein analysis systems. Separation of proteins is achieved by isoelectric focusing in a short length of fused-silica capillary after which the resolved proteins are immobilized to the capillary wall using photochemistry. The capillary is then evacuated, and fluorescently labeled antibodies are flowed through the capillary to bind to the immobilized proteins. This technique provides high sensitivity, the ability to spatially resolve and quantify proteins, and provides the opportunity for complete automation. Results obtained by fluorescence detection are compared to those obtained by chemiluminescence while offering enhanced resolution and signal stability. Capillary isoelectric focusing (cIEF) has been shown to be a powerful technique in its ability to resolve proteins.1-5 In many cases, only a short length of capillary is needed for sufficient resolution of the sample proteins, allowing the entire length of the capillary to be imaged, a process known as whole-column imaging detection (WCID). Such whole-capillary detection can significantly improve detection sensitivity over single-point detection methods.6-9 In this paper, we describe a fluorescence-based detector for full-span, high-resolution imaging of native and immunolabeled proteins immobilized in such a capillary. The use of capillaries in proteomic analysis offers numerous advantages over traditional slab gel-based separation methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).9,10 Capillaries can sequentially serve as the loading, separation, protein immobilization, reagent delivery, and detection devices, which greatly facilitates sample handling and automation. The internal volume of the capillary is very small, often only a few hundred nanoliters. This reduces sample and reagent con* To whom correspondence should be addressed. Phone: 1-(650) 859-1482. Fax: 1-(650) 845-3540. E-mail
[email protected]. (1) Wu, X. Z; Pawliszyn, J. Anal. Sci. 2001, 17, i189-i192. (2) Graf, M.; Watzig, H. Electrophoresis 2004, 25, 2959-2964. (3) Righetti, P. G.; Gelfi, C.; Conti, M. J. Chromatogr., B 1997, 699, 91-104. (4) Wu, J.; Pawliszyn, J. J. Chromatogr. 1992, 608, 121-130. (5) Wu, J.; Pawliszyn, J. Anal. Chem. 1992, 64, 2934-2941. (6) Wu, X.-Z.; Huang, T.; Mullett, W. M.; Yeung, J. M.; Pawliszyn, J. J. Microcolumn Sep. 2001, 13, 322-326. (7) Wu, X.-Z.; Pawliszyn, J. Electrophoresis 2002, 23, 542-549. (8) Beale, S. C.; Sudmeier, S. J. Anal. Chem. 1995, 67, 3367-3371. (9) Das, C.; Xia, Z.; Stoyanov, A.; Fan, Z. H. Inst. Sci. Technol. 2005, 33, 379389. (10) Ganzler, K.; Greve, K. S.; Cohen, A. S.; Karger, B. L.; Guttman, A.; Cooke, N. C. Anal. Chem. 1992, 64, 2665-2671.
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sumption and facilitates quantitative analysis. Three types of detectors have been used for WCID, namely, UV absorption, concentration gradient, and laser-induced fluorescence. UV absorption, while relatively simple, suffers from low sensitivity1,2,4,7,11 while concentration gradient has been shown to be more sensitive.4,5 Both UV absorption and concentration gradient detection, however, may have difficulty to reliably identify a protein of interest in a complex matrix such as a cell lysate. As such, these methods may be limited to analysis of highly purified protein samples. To selectively identify a protein of interest in such a complex mixture, it is often useful to introduce a second dimension of selectivity. One such method is to introduce the gene for a fluorescent protein onto the gene for the protein of interest.11,12 The protein of interest is then expressed with the fluorescent tag and may easily be identified in the complex mixture of the lysate. This method is limited in its usefulness to samples that can be transfected with the reporter fluorophore. Another method to identify proteins of interest is immunoblotting, a step critical to performing a conventional Western blot. In this procedure, proteins first separated on a polyacrylamide gel are transferred to a nitrocellulose membrane for immunolabeling.13 The addition of the immunoblot step dramatically increased the versatility of the SDS-PAGE procedure, and the Western blot remains one of the most important proteomic analytical techniques available to date. While assays involving the separation of immunolabeled proteins have been performed in capillary electrophoretic systems,14-18 such systems involve the formation of the immunocomplex prior to separation and are limited to the use of simple monoform ligands and separation conditions that favor proteinligand complexation. A more flexible approach is to immobilize the native proteins after separation. In a closed capillary, the transfer of the proteins to a separate membrane as is done in a Western blot is not feasible; however, immobilization can be achieved with the application of an UV activated coating to the inner wall of the capillary. Upon activation, the proteins are fixed (11) Hutterer, K.; Vladislav Dolnı´k, V. Electrophoresis 2003, 24, 3998-4012. (12) Korfa, G. M.; Landersb, J. P.; O’kanea, D. J. Anal. Biochem. 1997, 251, 210-218. (13) Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350-4354. (14) Shimura, K.; Karger, B. Anal. Chem. 1994, 66, 9-15. (15) Tao, L.; Kennedy, R. T. Anal. Chem. 1996, 68, 3899-3906. (16) Bornemann, C.; Burggraef, T.; Heimbu ¨ chel, G.; Hanisch, F.-G.; Winkels, S. Anal. Bioanal. Chem. 2003, 376, 1074-1080. (17) Cunliffe, J. M.; Liu, Z.; Pawliszyn, J.; Kennedy, R. T. Electrophoresis 2004, 25, 2319-2325. (18) Tan, W.; Fan, Z. H.; Qiu, C. X.; Ricco, A. J.; Gibbons, I. Electrophoresis 2002, 23, 3638-3645. 10.1021/ac071537z CCC: $37.00
© 2007 American Chemical Society Published on Web 11/17/2007
Figure 1. (a) Schematic representation of fluorescence imager. Three lasers are used, the first is a 488-nm argon ion laser set at 20 mW of emission power while the second laser is a frequency-doubled Nd:YAG with 10 mW of 532-nm light. The third laser is a 10-mW HeNe laser with 633-nm emission. Each beam is filtered and expanded with individual filters and beam expanders (B/E). A series of mirrors (M1-3) then redirect the beams into a pair of spherical lenses L1-2, which focus the beams onto the capillary. Light emission from the capillary is collected by a microscope objective L3, transmitted through an emission filter (filter 4), and refocused by L4 onto the slit, which selectively removes much of the off-axis light noise while transmitting most of the signal light to the PMT. (b) Detector optics. The excitation laser waist excites flurophores on the lumen of the capillary (large sunburst) while minimizing luminescent background from debris on the outer surface of the capillary. The light is collected by the microscope objective, filtered with a stacked pair of dichroic filters, and focused onto a slit by a second lens. The slit is positioned to pass light in the center while blocking light from sources such as scattering or fluorescence from dust (small sunburst) on the outer diameter of the capillary.
to the capillary wall, allowing the mobile contents of the capillary to be flushed out and pre-labeled immunoreagents flowed through the lumen.19 The capillary is then ready for scanning. For a system such as this where the analyte is present only in a thin film along the lumen wall a particularly sensitive detection method is required. For this we have developed a three color, laser based fluorescence scanner capable of detection along the entire length of the capillary. Fluorescence detection for capillary based systems offers several key advantages including excellent sensitivity, specificity, resolution, reagent availability, chemical stability, ease of use and the possibility of multiplex analysis. EXPERIMENTAL SECTION Assay Method. The instrumentation used for capillary isoelectric focusing, immunolabeling, and chemiluminescence detection is described in detail elsewhere;19 however, a brief overview is provided here to elucidate the process. The experiments are conducted in fused-silica capillaries 100 µm i.d. by 5 cm in length. The capillaries used in these experiments have a clear, Teflonbased external coating, which gives the capillary significant physical robustness without significant loss of transparency as compared to bare glass. The sample, typically a homogeneous mixture of proteins and a separation buffer system, initially fills the entire capillary. Fluorescent standards with known isoelectric (19) O’Neill, R. A.; Bhamidipati, A.; Bi, X.; Deb-Basu, D.; Cahill, L.; Ferrante, J.; Gentalen, E.; Glazer, M.; Gossett, J.; Hacker, K.; Kirby, C.; Knittle, J.; Loder, R.; Mastroieni, C.; MacLaren, M., Mills, T.; Nguyen, U.; Parker, N.; Rice, A.; Roach, D.; Suich, D.; Voehringer, D.; Voss, K.; Yang, J.; Yang, T.; Vander Horn P. B. Proc. Nat. Acad. Sci. U.S.A. 2006, 103, 16153-16158.
values are also included in the mixture. Protein separation is typically achieved in 4-30 min by isoelectric focusing (IEF) depending on the proteins being resolved. Immediately upon the completion of protein focusing, ultraviolet light is used to activate the benzophenone component of the inner coating. The excited benzophenone forms a diradical20 that may then form covalent bonds with the resolved proteins and the simultaneously resolved fluorescent peptides. This covalent attachment of the analyte proteins and fluorescent standards to the capillary wall leaves an open lumen through which the other reagents can be passed. After this immobilization, the following solutions are sequentially flowed through the capillary: wash solution, primary antibodies, wash solution, secondary antibodies, and wash solution. The secondary antibodies are typically prelabeled by the vendor with a fluorophore or an enzyme such as horseradish peroxidase (HRP), which is used as a catalyst in the chemiluminescent reaction.19 Each of these labels yields a spectrally unique signal from the flurophores of the peptides. The capillaries are then scanned using the fluorescence detector or the CCD camera based imager as described in ref 19. Apparatus. The key aspects of the fluorescence detector are illustrated in Figure 1. As can be seen in part a of the figure, the detector utilizes three lasers; an argon ion laser (JDS Uniphase 2214-20SL) with 20 mW of 488-nm emission, a frequency-doubled Nd:YAG laser (JDS Uniphase CDPS532M) with 10 mW of 532nm emission, and a helium-neon laser (Melles Griot 25 LHP 991) (20) Turro, N. J. Modern Molecular Photochemistry; University Science Books: Sausalito, CA, 1991.
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with 10 mW of 633-nm emission; however, only one laser is used for any given scan. The beams of each laser are first individually filtered and expanded using a laser line filter and a pair of cylindrical lenses. These lenses are used to shape each beam to an elliptical Gaussian profile. Each beam is then focused into the capillary by a spherical lens. A Newport PAC 055 f ) 120 mm is used to focus the 488and 633-nm beams, while a Newport PAC 052 f ) 100 mm lens is used for the 532-nm beam. At the focal points each beam measures ∼30 µm by 120 µm. The emission of the 488-nm laser is first transmitted through a Chroma z488/10× filter and expanded with a pair of cylindrical lenses (Newport CKX006 f ) 6.4 mm, Optosigma 022-0340 f ) 40 mm) for a total factor of 6.25×. The 532-nm beam is treated with a band-pass filter (Chroma HQ525/ 40) and expanded 3× using a Melles Griot 06GPA004 expander. The 633-nm beam is filtered with a band-pass filter (Semrock LL01-633) and expanded 5.95× with a beam expander created using a pair of cylindrical lenses (Newport CKX006 f ) 6.4 mm and Newport CKX038 f ) 38.1 mm). Each beam is then redirected by a series of mirrors (Newport 10D20BD.1) to the focusing lenses. The path taken by the 532-nm beam is unique while the path taken by the 488- and 633-nm beams is shared. This is achieved by the use of a mirror on a folding mirror mount (New Focus 9891). Each beam is then focused into the capillary by the aforementioned spherical lenses. Figure 1 shows the advantages of the elliptical beam profile used to illuminate the capillary. The 120-µm-long axis of the ellipse spans the 100-µm capillary lumen with an additional 10 µm per side. This allows the beam to efficiently excite the fluorophore labels despite any bow in the capillary while minimizing background caused by illumination of the outer wall. The 30-µm short beam waist along the separation dimension ensures excellent resolving power between protein bands separated along the capillary length. The capillary is placed on a cradle composed of a pair of notched knife edges attached to a computer-controlled motorized translation stage. This cradle precisely moves the capillary along its long axis, which is orthogonal to both the excitation laser beams and the microscope objective used as a collection lens (Meiji 50 × 0.5 NA). The collected fluorescent light is then transmitted through a pair of dichroic filters to reject any excitation light; the selection of these filters depends on the laser being used: For flurophores excited with the 488-nm laser, a Chroma Technologies HQ500LP filter and a HQ 525/40 filter are used. A pair of Chroma Technologies HQ580LP filters are used with the 532-nm laser, while a pair of Chroma Technologies HQ 700/75M filters are used with the 633-nm laser. The filtered light is then focused onto a narrow slit by a lens (Newport PAC 052 f ) 100 mm). The slit is shaped to pass light from the lumen of the capillary that contains the desired fluorescent signal while blocking the light from the outer portions of the capillary that contain undesired background light. The filtered signal continues to the photomultiplier tube (PMT; Hamamatsu R3896) operating at a bias of 500 V. The electrical signal from the PMT is amplified using a Stanford Research Systems SR570 preamplifier. The amplified signal is digitized with a PC board-based, 16-bit A/D converter (National Instruments NI PCI 6014B series) and then stored using PC software designed in-house. 9480
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Samples. Recombinant green fluorescent protein (rGFP; Clontech 632373) was chosen as the analyte for these experiments. This protein possesses many attractive characteristics including excellent excitation overlap with the 488-nm line of the argon laser of the setup, high native fluorescence yield, good stability, and the ready availability of anti-GFP antibodies. The native fluorescence is particularly attractive as this allows a direct comparison of results obtained by immunolabeling to that of the protein alone. The rGFP stock used in these experiments was prepared by adding 10 µL of 1 mg/mL rGFP to 46 µL ddH2O, 25 µL of 80% sorbitol, and 15 µL of NDSB 256 (D465250; Toronto Research Chemical, NY, ON, Canada), and 5 µL of broad-range ampholytes, pI 3-10 (Sigma 1522). Also added to the mixture was 2 µL each of in-house-generated fluorescent peptide standards of pI 4.92 and 7.01, respectively. The final concentration of rGFP in the stock solution is 100 µg/mL. ddH2O was substituted for the rGFP in dilutent and control solutions. Fluorescent Peptide Standards. Fluorescent peptide standards of known isoelectric point were made by standard peptide synthesis protocols using an ABI 433A peptide synthesizer and Fmoc chemistry.19 The sequences of the fluorescence peptide standards used are as follows: pI 4.92 standard, H2N-K(ATFB)GaehehehekeK(5-TAMRA)G-OH; pI 7.01 standard, H2N-K(ATFB)GaehrK(5-TAMRA)G-OH. Upper case character denotes an L-amino acid, and lower case letter denotes a D-amino acid. ATFB is 4-azido-2,3,5,6-tetrafluorobenzoic acid and 5-TAMRA is 5-carboxytetramethylrhodamine. The isoelectric point of each standard was determined by measuring the pH of the gel in the region of each focused band in a conventional IEF gel as described by Shimura et al.21 Capillary Preparation. Two-meter lengths of Teflon-coated fused-silica capillaries with interior vinyl coating (100 µm i.d. × 375 µm o.d. product number TSU 100375, Polymicro Technologies, Phoenix, AZ) were cut from a 50-m-length stock. These sections then had polyacrylamide copolymerized with 4 mol % acrylbenzophenone monomers grafted onto the lumen walls.19 The capillaries were then cleaved into 5-cm sections and used as described below. Isoelectric Focusing and Immobilization. Capillaries were loaded by immersing the capillary tip in the sample solution and applying enough vacuum to ensure complete filling of the capillary. Separation and immobilization of rGFP samples was performed by placing the capillaries horizontally in the holder described in ref 19. In this holder, the capillary tips were immersed in reservoirs containing 10 mM H3PO4, pH 2, with 1% w/v poly(ethylene oxide) (PEO) in the anodic end and 100 mM NaOH, pH 12, containing 1% w/v PEO in the cathodic end. The presence of PEO minimizes any hydrodynamic flow that may result from small fluid height differences between capillary ends. To accomplish isoelectric focusing of GFP samples, typically an initial potential of 750 V was applied for 400 s, followed by 1500 V for 700 s. Upon completion of the focusing step, the highintensity UV lamp was activated for 15-60 s to initiate the crosslinking process and immobilize the proteins and peptide standards. After immobilization, the capillaries were removed from the capillary holder and the anodic end of each capillary was placed (21) Shimura, K.; Kamiya, K.; Matsumoto, H.; Kasai, K. Anal. Chem. 2002, 74, 1046-1053.
in contact with a TBST solution consisting of 10 mM Tris-HCl, 150 mM NaCl and 0.05% Tween 20 at pH 6.8. A 5-mmHg vacuum source was applied to the cathodic end of each capillary, and the TBST solution was continuously pulled through each capillary for 20-60 s. Washing and Antibody Probing, Unlabeled polyclonal rabbit/R-GFP IgG fraction primary antibody (Invitrogen A11122) was used with either an Alexa 647-labeled goat/R-rabbit IgG (H+L) secondary antibody (Invitrogen 21244) or a HRP-labeled goat/Rrabbit IgG (H+L) conjugate secondary antibody (Zymed 81-6120). A portion of the Zymed secondary antibody was also colabeled with Alexa 647 fluorophore using an antibody labeling kit (Invitrogen A-20186). The immunoassay was performed using the apparatus described in ref 19. A 5-mmHg vacuum source was used to pull TBST buffer through the capillaries for 4 s. The vacuum was then stopped, and the capillaries were allowed to rest for 15 s. This was repeated 10 times. After this wash step, the capillary tips were then transferred to a solution of the primary antibody diluted 500fold from stock in TBST for a final concentration of 4 µg/mL. This solution was flowed through the capillaries using the same vacuum source for 3 s followed by a 120-s incubation time with no vacuum. This was also repeated 10 times. Following this primary immunolabeling step, the capillaries were again washed using the same protocol as the initial wash step. The capillaries were then ready for the second immunolabeling step. In this step, a solution containing one of the secondary antibodies diluted by a factor of 1000 in TBST (final concentration of 2 µg/mL) was sequentially flowed through the capillary and incubated as in the primary immunolabeling step, the specific antibody varied with the experiment being performed. Finally, after the completion of the second immunolabeling step, the capillaries were given a final wash following the same protocol as the previous washes. Fluorescence Detection. Capillaries were placed one at a time in the cradle of the detector and scanned at 0.1 mm/s at a sampling rate of 50 Hz. For measurement of native GFP fluorescence, the scanner was configured for use with the 488-nm laser. Emitted fluorescence was passed through a pair of filters to yield a 525 ( 20 nm emission band-pass filter. The peptide standards were measured with the scanner configured for the 532-nm laser with the emitted light passed through the 580-nm emission longpass filters while the fluorescence signal from the Alexa 647/HRP dual-labeled antibody was measured using the scanner configured for the 633-nm laser and the 700 ( 38 nm emission band-pass filters. Chemiluminescence Imaging. Capillaries imaged by chemiluminescence were placed in the CCD camera-based detector.19 A mixture of equal parts SuperSignal West Femto Stable Peroxide buffer and Luminol/Enhancer solution from Pierce (Rockford, IL, part numbers 1859023 and 1859022, respectively) was pulled through the capillaries with 5 Torr of vacuum. Capillaries were placed horizontally in the capillary holder, and a slight excess of the above chemiluminescence reagent mix was supplied to one of the two reservoirs to create a slight hydrostatic head and resultant continuous flow of reagent. Chemiluminescence signal was collected for 10 s to 10 min depending on signal strength.
Figure 2. Sample data for determination of percent capture of protein. Peak in large graph represents fluorescence trace for unbound GFP hydrodynamically shifted away from the area of immobilized protein. The inset is an enlargement of the trace of the immobilized protein, which shows three peaks corresponding to the observed isoforms of GFP. From the ratios of the areas of the immobilized protein trace and the unbound GFP protein, the percent of protein immobilized is estimated to be 0.032%.
Data and Statistical Analysis. The fluorescence data were imported into IGOR Pro v5.03 (Wavemetrics, Lake Oswego, OR) software for filtering and graphing. Upon importation, the data were treated with a low-pass filter to remove high-frequency background noise. The chemiluminescent data were treated somewhat differently. One-dimensional isoelectropherograms were extracted from the raw camera TIFF using a program derived in-house and exported to IGOR. The resulting fluorescence and chemiluminescent electropherograms were then analyzed for peak positions, widths, and heights. RESULTS Capture Dynamics. The dynamics of protein capture on sample concentration was examined using two separate experiments. In the first, a serial dilution series of rGFP protein was focused, captured, and immunolabeled as described above. The capillaries were then scanned using the fluorescence scanner configured for detection of native GFP fluorescence. After the fluorescence detection step, the capillaries were imaged using chemiluminescence. As previously reported, this combination of detection techniques showed protein immobilization to be directly proportional to protein concentration over more than 6 orders of magnitude.19 In a second series of experiments, an individual capillary was focused and captured as before but the immunolabeling step was not performed. Instead, the unbound GFP protein remaining in the capillary was hydrodynamically shifted away from the point of immobilization, spatially separating the bound and unbound protein. The capillary was then immediately scanned for native GFP fluorescence to minimize diffusion of the unbound protein. Sample results from this procedure are shown on Figure 2. Analytical Chemistry, Vol. 79, No. 24, December 15, 2007
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Table 1. Peak Area Ratios of Immobilized and Unbound GFP in Six Capillariesa
a
trial
[GFP], µg/mL
peak area ratios, %
1 2 3 4 5 6
20 10 5 20 10 5
0.032 0.030 0.031 0.032 0.032 0.038
Average capture efficiency is 0.032%.
The large image shows both the immobilized and unbound protein while the insert shows an enlargement of the immobilized protein only. By comparing the area beneath the peaks corresponding to immobilized and unbound proteins, the average capture efficiency for GFP is estimated to be 0.032%. Table 1 shows the data for these measurements. Experiments such as this highlight the flexibility of fluorescent based WCID for its ability to measure both the immobilized and free protein. Limit of Detection. The limit of detection of GFP using native fluorescence detection on the fluorescence scanner is estimated to be 40 pM while the limit of detection for fluorescently immunolabeled GFP on the fluorescence scanner is estimated to be 1 pM using the Invitrogen Alexa 647-labeled antibody. The limit of detection using the chemiluminesent imaging system is estimated to be 300 fM using GFP immunolabeled with the Zymed HRP antibody. Each value is estimated to a signal-to-noise ratio of 3, where the signal is defined as the height of the signal peak and the noise is defined as the standard deviation of the baseline. Given the probable variance in binding affinity of the antibodies as well as the average number of flurophore or enzyme labels per antibody, there does not appear to be a clear advantage in the detection limit of one method over another. Resolution. One area where fluorescence does show an advantage is peak resolution. A comparison of isoelectrically focused GFP detected by chemiluminescence, native fluorescence as well as immunofluorescence is presented in Figure 3. All traces shown in the figure were obtained from the same protein sample in the same capillary. Trace a is the profile obtained from the native fluorescence of the sample. Trace b is the fluorescence signal obtained from the Alexa 647 flurophore on the antibody (the spectral cross-talk from the native fluorescence signal was found to be negligible in this channel). Trace c is the chemiluminescent signal obtained from the HRP label on the same antibody. As can be seen in the graph, the fluorescent signal from the antibody reflects the profile of the native fluorescence signal while the profile of the chemiluminescent trace is significantly broadened. Table 2 gives the statistical data for four capillaries. Using eq 1, it is determined that on average a peak detected using chemiluminescence will on average be broadened by 166 µm ( 67 µm n
∑ xfwhm
fwhmBB )
2 C
n)1
n
- fwhmF2 (1)
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Figure 3. Comparison of fluorescence and chemiluminescence detection of cIEF-separated GFP. (a) GFP imaged using native fluorescence, (b) GFP detected with labeled antibody based fluorescence, and (c) GFP detected with labeled antibody-based chemiluminescence. Table 2. Fluorescence and Chemiluminescence Immunolabeling Data for Four Replicates of rGFP Analyzed by cIEFa fwhm (µm) cap 1 2 3 4
average standard deviation
isoform peak
Fluor Alexa 647
Chemi HRP
1 2 3 1 2 3 1 2 3 1 2 3
152 142 148 148 170 184 170 156 148 174 166 148
182 250 227 182 318 227 227 318 227 182 273 205
159 13
235 48
a Peak data correspond to each of the three observed isoform peaks for GFP.
where fwhmC is the full width half-maximum of the peak as detected by chemiluminescence and fwhmF is the same peak detected by fluorescence. This broadeningsfwhmBBsis assumed to be due to the diffusion of the activated enhancer prior to emission (assuming Gaussian peak profiles). Chemiluminescence Signal Drop. An additional issue with chemiluminescence detection is the irreversible decrease of the signal intensity with time. This effect can be observed in Figure 4. This figure shows a test capillary containing focused GFP after chemiluminescent immunolabeling. Part a of the figure shows two traces; the upper trace shows the initial native fluorescence signal just prior to the introduction of the luminol while the lower trace shows the native fluorescence signal from the same capillary after having luminol reagent flow through it overnight. Part b of the
both cases, the signals were completely lost by the second day. Control capillaries not exposed to luminol did not show loss of native fluorescence signal (data not shown). The loss of native fluorescence and chemiluminescent signals does not seem to be solely the result of decoupling of the proteins from the capillary wall but instead may also be the result of protein denaturation or partial degradation of their primary structures. Denatured GFP is known to be nonfluorescent22,23 and denaturing; in addition to degradation by the luminol reagent of this protein-based fluorophore, the antibodies and their HRP labels may be one potential cause for the loss of both the native fluorescence and chemiluminescence. To test this hypothesis, the capillary was then subjected to a second cycle of immunolabeling and scanned. Figure 4b shows the chemiluminescent signal obtained from the same capillary after this second cycle of immunolabeling. As can be seen from the figure, a portion of the original signal is regained indicating at least a portion of the captured GFP protein remains on the capillary walls and available for immunolabeling with the polyclonal primary antibody.
Figure 4. (a) Native fluorescence and (b) chemiluminescence profiles of HRP-immunolabeled GFP. The initial native fluorescence signal was measured just prior to the introduction of the luminol reagent while the initial chemiluminescence signal was measured just after the introduction of the luminol (top traces). Luminol reagent was then flowed through the capillary overnight and the capillary reimaged the next morning (middle traces). Finally, the capillary was subjected to a second round of immunolabeling and imaged again for partial restoration of chemiluminescent signal (bottom traces).
figure shows the chemiluminescent data from the same test capillary with the upper trace showing the data obtained just after the introduction of luminol and the lower trace showing the data obtained after capillary was flushed overnight with luminiol. In (22) Cody, C. W.; Prasher, D. C.; Westler, W. M.; Prendergast, F. G.; Ward, W. W. Biochemistry 1993, 32, 1212-1218. (23) Chalfie, M.; Tu, Y.; Euskrichen, G.; Ward, W. W.; Prasher, D. C. Science 1994, 263, 802-805.
CONCLUSION Capillary-based proteomic analysis offers many advantages over traditional, slab gel-based analytical methods. Lower sample consumption, higher throughput, greater resolution, and fully automated analysis are just a few of the benefits. There are, however, some inherent challenges to such a system, e.g., the low overall signal levels and minute volumes presented by capillaries. In this paper, we have presented a fluorescence-based scanner that offers advantages over chemiluminescence for detection of immobilized proteins in a capillary electrophoretic system. Ease of use, better resolution, comparable detection limits, and chemical stability make fluorescence an attractive solution for such a system. Fluorescence-based detection also allows the possibility of multiplexing through the use of multiple antibodies labeled with unique fluorophores. Received for review July 20, 2007. Accepted September 28, 2007. AC071537Z
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