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Anal. Chem. 1986, 58,506-507
Sensitive Photothermal Densitometer for Quantitation of Coomassie Brilliant Blue Stained Proteins in Polyacrylamide Gels Konan Peck a n d Michael D. Morris* Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109 Photothermal techniques are being developed for the detection of molecules eluting from liquid chromatographs. The principal advantages are the ability to make measurements with microabsorbance noise levels in volumes of a few tens of nanoliters. The technique used most commonly has been thermal lens spectrometry (1-4), although a related technique, photothermal deflection, has been applied to the problem (5). Photothermal deflection has been explored for quantitation of molecules separated by thin-layer chromatography (6-9). Here, too, greater sensitivity than can be obtained by conventional densitometry has been reported. Electrophoresis is as central to the study of charged polymeric molecules, principally proteins and nucleic acids, as chromatography is to the separation of many other kinds of molecules. Electrophoretic separations sort charged macromolecules by size, molecular weight, secondary structure, and electric charge. Laboratories devoted to the study of these biopolymers typically employ electrophoresis at each characterization or fractionation step. Proteins and nucleic acid are made up of relatively few different kinds of primary structural units. These are transparent in the visible and much of the ultraviolet. The natural absorbance of amino acids, purines or pyrimidines, is, in any case, not very high in the accessible ultraviolet. Thus, the progress of electrophoresis has included increasingly sensitive methods for visualization of proteins and amino acids. Throughout much of the 19609 and 19709 proteins separated by electrophoresis were visualized by staining with Coomassie Brilliant Blue ( l O - l Z ) , which provided a 10-fold increase in sensitivity over the dyes that it replaced. In the last few years Coomassie Brilliant Blue staining has been displaced in many applications by silver staining. In this technique the polyacrylamide gel is soaked with silver nitrate, and the ionic silver is reduced to metallic silver. Silver staining provides a factor of 50-100 increase in sensitivity over Coomassie Brilliant Blue (13-15). However, silver staining is a more difficult and demanding technique than Coomassie Brilliant Blue staining. Moreover, no staining technique, including silver staining, is applicable to all proteins. There remains a need for simple and sensitive techniques for quantitation of different stains. EXPERIMENTAL SECTION Acrylamide and N,”-methylenebis(acry1amide) (Bis) (Sigma) were purified by Loening’s recrystallization procedure (16). Coomassie Brilliant Blue G250, ammonium persulfate, bromphenol blue, tetramethylethylenediamine (TEMED), and bovine serum albumin (BSA) (Sigma) were used as received. All other chemicals were ACS reagent grade and were used without further purification. Type-I deionized water was used to prepare all gels and solutions. Solutions were passed through a 0.45-pm membrane filter before use. Polyacrylamide gels were prepared according to Davis (17). Sodium dodecyl sulfate (SDS) polyacrylamide gels were prepared by the procedure of Laemmli (18). Electrophoresis was carried out on a locally constructed vertical slab (Studier) chamber at room temperature. Gels were streaked with sample in 10-mm channels. After electrophoresis,gels were stained by gentle agitation in Coomassie Brilliant Blue (2250 (19) and then destained in 5% acetic acid to minimize background. The stained gels were then cut into 25 X 100 mm strips, each containing two channels, and placed between quartz plates in a locally constructed holder.
The thermal lens densitometer is shown in Figure 1. The design is based on the single laser/dual crossed beam principle (20). Reproducible crossing angles were obtained by using the aluminized prism apparatus previously described (21). A He-Ne laser (Spectra-Physics 124) providing 40 mW at the laser head was used as the light source and yielded 20 mW heating power at the sample. The heating beam was modulated with a mechanical chopper (Laser Precision CTX-534) at 7.4 Hz. The thermal lens signal was detected by allowing the probe laser to overflow an adjustable iris. The light passing this limiting aperture was sensed with a photodiode, whose output was demodulated with a lock-in amplifier (PARC 5101) operated with a 1-9 time constant. The demodulated signal was sampled with a 12-bita-d converter and stored on a small computer. Electrophoresis gels were scanned by moving the gel holder through the intersecting laser beams at 17 mm/min. Peak area was taken as a measure of thermal lens response. For display purposes records have been smoothed, although all calculations are based on the raw data as recorded. RESULTS AND DISCUSSION Figure 2 shows the polyacrylamide gel electrophoretograms of bovine serum albumin a t quantities ranging from 3 to 60 ng. The albumin peak occurs approximately 4.2 cm from the anode of the apparatus. Albumin samples smaller than 125 ng do not yield visible stains. Even so, easily measured peaks are obtainable from nanogram quantities of albumin. Because of the high sensitivity of the technique, dust particles, air bubbles, scratches on the gels, and minute quantities of trace proteins on the track of protein migration are all detectable by the thermal lens method. Dust, air bubbles, and scratches scatter the probe beam and result in characteristically sharp spikes. These are easily distinguished from real protein/stain thermal lens signals by their sharpness. Traces of protein impurities along the scan axis produce the wavy base line and offset visible in the scans of small quantities of albumin. Contamination can be minimized by careful sample handling. It is likely that further background reduction is possible if albumin purified by several recrystallizations is used. A second band (not shown) for albumin dimer appears farther from the anode, out of the scan range of our apparatus. Denaturing the proteins into their subunits with SDS yields a single albumin peak. However, SDS-polyacrylamide gel electrophoresis produces higher backgrounds, because SDS is also stained by Coomassie Brilliant Blue G250 (22). Trace amounts of SDS are always detectable by the thermal lens method. Preliminary results suggest that a better dye for SDS-gel electrophoresis may be Coomassie Brilliant Blue R250, if extensive destaining is employed. At least 2 days of diffusional destaining is necessary to remove background (23). Our laboratory is not equipped for electrophoretic destaining, which could reduce the required time to less than 1 h. Therefore, we have conducted the bulk of our experiments using the Davis procedure (17)rather than the more commonly encountered Laemmli technique (18). Peak areas under the albumin band increase linearly with albumin mass up to at least 60 ng. Above 100 ng the slope of the calibration curve begins to decrease. The detection limit, take as S I N = 2, is 0.95 ng. By use of the full width half-height of a 3-ng peak as a measure of protein band width,
0003-2700/86/0356-0506$01.50/00 1986 American Chemical Society
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Anal. Chem. 1986, 58,507-509 Lictr
Ha-Ne
Photodiode
Figure 1. Schematic diagram of the thermal lens densitometer: focusing lens 200 mm focal length; lens after the iris 100 mm focal
length.
k
56 z
tometer light source (25).The focused diameter is 0.061 mm, so excellent spatial resolution should be obtainable with minor modification of our instrument. Because the thermal lens densitometer detects the protein indirectly by measuring the binding dye, detection limits might be further reducted by more sensitive staining methods, such as silver staining. Nevertheless the enhancement of the sensitivity of Coomassie Brilliant Blue staining by thermal lens densitometer already provides clear advantages. First, the sensitivity of the simple Coomassie Brilliant Blue staining method can be made to match the sensitivity of the more complicated silver staining procedure, as measured with conventional instruments. Second, thermal lens detection of Coomassie Brilliant Blue stains provides senitive quantitation for proteins not amenable to silver staining (26-31). Extension of the thermal lens technique to quantitation of silver stains and exploration of other proteins is currently in progress. Registry No. Coomassie Brilliant Blue, 6104-58-1.
LITERATURE CITED (1) Sepanlak, M. J.; Vargo, J. D.; Kettler, C. N.; Maskarinec, M. P. Anal. Chem. 1984, 56, 1252-1257. (2) Leach, R. A,; Harris, J. M. J . Chromatogr. 1984, 218, 15-19. (3) Buffett, C. E.; Morrls, M. D. Anal. Chem. 1992, 54, 1174-1178. (4) Pang, T.-K. J.; Morris, M. D. Anal. Chem. 1984, 56, 1457-1459. (5) Collete, T. W.; Parekh, N. J.; Men, L. C.; Carreria, L. A,; Rogers, L. B. Pittsburgh Conference Abstract, 1985;056. (6) Chen, T. I.; Morris, M. D. Anal. Chem. 1984, 56, 19-21. (7) Chen, T. 1.; Morris, M. D. Anal. Chem. 1984, 56, 1674-1677. (8) Masujima, T.; Sharda, A. N.; Lloyd, L. B.; Harris, J. M.; Eyring, E. M. Anal. Chem. 1984, 5 6 , 2977-2979. (9) Peck, K.; Fotlou, K. F.; Morris, M. D. Anal. Chem. 1985, 5 7 ,
1359-1362. (IO) Fishbein, W. N. Anal. Biochem. 1972, 46, 388-401. (11) Fazekas de St. Groth, S.; Webster, R. G.;Datyner, A. Blochim. Biophys. Acta 1983, 31, 377-391. 3
4
5
6
DISTANCE FROM ANODE (cm)
Figure 2. Electrophoretogram scanned by the thermal lens technique:
nondenaturing discontinuous electrophoresis of bovine serum albumin on 10% polyacrylamide gels with quantities as shown; insert, top to bottom, 6 ng, 4 ng, 3 ng of bovine serum albumin. The albumin dimer band is not shown. the detection limit is area normalized to approximately 45 pg/mm2. This detection limit is similar to the silver-staining limit, reported as 20 pg/mm2 (24)to 42 pg/mm2 (15)in recent studies.
CONCLUSIONS We confirm the previous observations (19)that quantities of Coomassie Brilliant Blue stained bovine serum albumin less than about 125 ng are not visible to the eye. Nevertheless thermal lens densitometry performs well with these quantities of albumin. The dynamic range of the photothermal densitometer can be extended to higher concentrations than those studied here. Because of the nonlinear response of the Coomassie Brilliant Blue dye bipding and the saturation of thermal lens signals a t high absorbances, the useful range may not extend above a few micrograms. However, in the microgram region, the laser can be used directly as a conventional transmission densi-
(1s) Chrambach, A.; Relsfeld, R. A.; Wyckoff, M.; Zaccari, J. Anal. Biochem. 1967, 20, 150-154. (13) Switzer, R. C., 111; Merril, C. R.; Shifrin, S.Anal. Blochem. 1979, 98, 231-237. (14) Oakley, B. R.; Kirsch, D. R.; Morris, N. R. Anal. Blochem. 1980, 105, 361-363. (15) Morrlssey, J. H. Anal. Biochem. 1981, 117, 307-310. (16) Loening, U. E. Biochem. J. 1987, 102, 251-256. (17) Davis, B. T. Ann. N. Y. Acad. Sci. 1964, 121, 404. (18) Laemmli, U. K. Nature (London) 1970, 227, 680-685. (19) Reisner, A. H.; Nemes, P.; Bucholtz. C. Anal. Biochem. 1975, 64, 509-51 6. (20) Yang. Yen.; Hairrell, R. E. Anal. Chem. 1964, 56, 3002-3004. (21) Wallan. D. J. Ritz, G. P.; Morris, M. D. Appl. Spectrosc. 1977, 37, 475. (22) Sedmak, J. J.; Grossberg, S. E. Anal. Biochem. 1977, 79, 544-552. (23) Wilson, C. M. Anal. Biochem. 1979, 96, 263-278. (24) Merrll, C. R.; Goldman, D.; Van Keuren, M. L. Eiectrophoresis (Weinhelm, Fed. Repub. Ger.) 1982, 3 , 17. (25) Brayden, J. E.: Halpern, W. Anal. Biochem. 1983, 130, 9-13. (26) Wray, W.; Boulikas, T.; Wray, V. P.; Hancock, R. Anal. Biochem. 1981, 116, 197-203. (27) Friedman, R. D. Anal. Biochem. 1982, 126, 346-349. (28) Schleicher, M.; Watterson, D. M. Anal. Biochem. 1983, 731, 312-31 7. (29) Poehling, H.-M.; Neuhoff, V. Nectrophoresis (Weinheim, Fed. Repub, Ger) 1981, 2 , 141-147. (30) Guevara, J.; Johnston, D. A.; Ramagali, L. S.; Martin, B. A,; Capetillo, S.; Rodriguez, L. V. Electrophoresis (Weinheim, Fed. Repub. Ger .) 1982, 3 , 197-205. (31) Tsai, C.-M.; Frasch, C. E. Anal. Biochem. 1982, 119, 115-119.
RECEIVED for review July 17, 1985. Accepted September 19, 1985.
Versatile Instrument for Pulse Width Measurement Purnendu K. Dasgupta* and Ellis L. Loree Department of Chemistry, Texas Tech University, Lubbock, Texas 79409-4260
A variety of devices are available for measurement of fast pulse widths, oscilloscopes being the most common instruments used for measurements of pulse width
