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A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration
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A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration
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Kevin R. Rupprecht ⇑, Ewa Z. Lang, Svetoslava D. Gregory, Janet M. Bergsma, Tracey D. Rae, Jeffrey R. Fishpaugh Diagnostics Analytical Chemistry Research, Abbott Laboratories, Abbott Park, IL 60049, USA
a r t i c l e
i n f o
Article history: Received 9 February 2015 Received in revised form 15 May 2015 Accepted 6 June 2015 Available online xxxx Keywords: Sodium dodecyl sulfate (SDS) Concentration Quantitative analysis
a b s t r a c t Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure). The improved method is simple to implement, robust, accurate, and (most important) precise. Ó 2015 Elsevier Inc. All rights reserved.
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Sodium dodecyl sulfate (SDS)1 is a widely used detergent in molecular biology and biochemistry, including protein purification [1–3] and analysis [4–8]. Its utility is offset somewhat because it is difficult to remove from the protein once added to a solution [9– 14]. Quantification of SDS concentration in protein solutions is of interest, yet few quantitative assays are both practical and precise. Several methods are known, but none has been widely adopted [15–22]. One method, although both accurate and precise, relies on the separation of SDS into an organic phase [15]. This method is time-consuming and requires extensive extraction with chloroform. The most attractive spectroscopic method uses the interaction between SDS and a dye (Stains-All), where the method is easy to perform and has a broad dynamic range of 0.35 to 70 mM (0.01–2.0% SDS) [20]. An additional advantage is that the authors also demonstrated that a large number of potential sample matrix components, including protein and nucleic acids that are known to be stained by Stains-All, had no effect on the absorption at 438 nm, the recommended wavelength for analysis. Unfortunately, we obtained coefficients of variation (CVs) of 10 to 30% using this method and needed much better precision for our use. This article describes a modification of the Stains-All method with improved precision, mitigation of dye solubility issues, reduced background, and reduced sample buffer matrix effects. The method has a dynamic range of 0.001 to 0.01% ⇑ Corresponding author. Fax: +1 224 668 3271. E-mail address: [email protected] (K.R. Rupprecht). 1 Abbreviations used: SDS, sodium dodecyl sulfate; CV, coefficient of variation; LOD, limit of detection; LOQ, limit of quantitation; DMF, N,N-dimethylformamide; AU, absorption units.
SDS, making it usable with a single dilution for protein solutions with SDS concentrations of 0.05 to 1.0%. Precision is generally high (1–5%CV), with defined limits of detection (LOD) and quantitation (LOQ) of SDS. A detailed examination of the protocol [20] revealed several areas for potential improvement. The dye stock solution was problematic because significant amounts of undissolved dye were observed in multiple preparations of stock solution. The solution was filtered; however, this led to an unknown final concentration of the dye and differing adsorption peak intensities, and the dye continued to precipitate over time at 2 to 8 °C. An alternative stock solution using N,N-dimethylformamide (DMF) dissolved the dye completely, consistently, and at dye concentrations greater than 2 mg/ml. The subsequent working solution originally included the solvent formamide, but we found that by using the above DMF stock solution, a working solution could be prepared by simple dilution with water. Effects of buffers on the dye spectra were also investigated. A buffer of 10 mM sodium phosphate and 15 mM NaCl at pH 7.2 was eventually chosen because it gave the best combination of absorbance and separation of wavelengths between SDS-bound Stains-All and unbound dye (see the online Supplementary material for additional discussion of the effects of various buffers). In addition, the wavelength previously used for quantification, 438 nm [20], was found to be on the side of the absorbance peak rather than at the apex (see Fig. 1), and the kmax varied from 445 to 460 nm. The steepness of the peak slope combined with the varying kmax led to poorer precision in our experience than reported by the original assay authors [20] and,
http://dx.doi.org/10.1016/j.ab.2015.06.013 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.R. Rupprecht et al., A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.06.013
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No. of Pages 3, Model 5G
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Notes & Tips / Anal. Biochem. xxx (2015) xxx–xxx
Fig.1. Spectra of SDS bound to Stains-All. Absorbance spectra of Stains-All with bound SDS solutions at 0, 0.001, 0.0025, 0.005, 0.0075, and 0.01% SDS are shown, where kmax (453 nm) is indicated as well as the wavelength previously used for measuring SDS concentration (438 nm) [20]. The spectra indicate that 438 nm is not optimal for precision. The data for these solutions were plotted to demonstrate assay linearity (R = 0.998).
86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
subsequently, to higher variation in SDS concentration. We used the A453 for quantitation to improve precision in our assay, and CVs decreased from P30 to 65.4%. Dyes can be photosensitive, and for our initial solutions there was an overall loss of more than 50% absorbance within 1 day, and the sensitivity to light is exacerbated with high concentrations of protein (100 lg/ml) in the final assay solution. The previous work [20] demonstrated that protein at a low concentration (4.33 lg/ml) in the final assay solution did not affect the absorbance of the SDS–Stains-All complex. Our sample preparation dilution yielded protein concentrations of less than 2 lg/ml in the final assay solution, and light-sensitive signal loss is minimized to less than 5%. The detailed protocol can be found in the Supplementary material. Our above modifications of the previous method [20] were developed using a cuvette assay measured with a spectrophotometer (macro format), and we adapted these modifications to a microtiter format. Depending on the microtiter system used, instrumentation limits may require a shift in the wavelength to 450 nm when using filter-based instruments, such as the ELx 808 Ultra Microplate Reader, that use a filter with a bandwidth of 440 to 460 nm. The data below were obtained using the ELx 808 Ultra Microplate Reader. Both the cuvette and the microtiter assays were designed to provide analysis of samples in the same concentration range, with similar assay performance characteristics. Assay performance was examined by linear fit, precision, reproducibility, LOD, and LOQ using the standards that ranged from 0.001 to 0.01% with water as a blank. The concentration of SDS was found to be highly linear over this range in both the cuvette and microtiter assays (A450 and A453). Standards were run in triplicate, with R values of 0.989 to 0.999 for the cuvette assay and 0.998 to 0.999 for the microtiter assay. Percentage CVs were determined for triplicate analyses to assess within-assay precision and were found to be lower for the microtiter assay (see Table 1; see also Table S2 in the Supplementary material). The lowest calibrator (0.001%) had CVs of 5.4% (cuvette) and 4.7% (microtiter), whereas the highest calibrator (0.01%) had CVs of 2.5 and 1.5%, respectively. Two types of control samples were used to aid in assessing performance; one set had concentrations of SDS in water (0.0033 and 0.0066%), and a second consisted of two protein solutions containing SDS (0.15 and 0.5%). The proteins used were proprietary
Table 1 Comparison of assay precision and reproducibility between the cuvette assay measured at 453 nm and the microtiter assay measured at 450 nm. Control
n
Cuvette average value
Cuvette average %CV
Microtiter average value
Microtiter average %CV
Control A (0.0033% SDS) Control B (0.0067% SDS) Sample control A Sample control B Linearity (R)
8
0.0035
3.46
0.0033
2.05
8
0.0074
2.98
0.0065
1.31
8 8 8
0.1491 0.4993 0.9964
2.46 3.68 0.69
0.1635 0.4857 0.9987
1.48 1.40 0.08
recombinant proteins having molecular masses of approximately 46 and 23 kDa. The microtiter assay again gave tighter CVs than the cuvette assay format, most likely due to the improved precision in pipetting the components. In the cuvette assay, the average CVs for the two aqueous SDS controls were 3.5 and 3.0%, with 2.5 and 3.7% for the two protein–SDS controls. In the microtiter assay, we observed average CVs of 2.1 and 1.3% for the aqueous SDS controls, with 1.5 and 1.4% for the two protein–SDS controls. We also compared the filter-based reader with a monochromator-based system, the Synergy Mx. The two microtiter systems produced highly comparable results (see Table S3). Reproducibility was examined in-depth using the microtiter format with three different lots of Stains-All purchased over a 3-year time range to determine lot-to-lot precision and consistency of the SDS–Stains-All interaction. All of the lots examined gave good precision, with CVs ranging from 0.27 to 3.42% for the standard solutions. The A450 absorbance of the high controls analyzed using the three Stains-All lots varied from the mean of the three lots by 0.012 to 0.026 absorption units (AU) (1.162 AU mean) and 0.006 to 0.011 AU (0.6336 AU mean) for the low control lots. Final SDS concentrations for all samples and controls varied from each other by ±3.1% for all three dye lots tested. Operator-to-operator variability was examined and produced CVs of less than 5.3% for all samples, controls, and standards, with the highest CVs seen with the lowest standard. Between-operator comparisons showed CVs of less than 4.9% for all samples and
Please cite this article in press as: K.R. Rupprecht et al., A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.06.013
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controls. Finally, a series of assays was used to determine LODs and LOQs in both the cuvette and microtiter assays. LOD was taken as 3 noise, with noise determined as the greatest difference observed between 24 blank determinations, and 10 noise was used to determine LOQ. For the cuvette assay, an LOD of 0.0004% SDS and an LOQ of 0.0008% SDS was achieved, whereas the LOD and LOQ for the microtiter assay were 0.0006 and 0.0008% SDS, respectively. The higher LOD for the microtiter assay reflects the shorter AU range achievable in the microtiter reader due to the shorter path length relative to the spectrophotometer used in the cuvette assay. Overall, both assays produced superior performance to other published methods and can be used for most biological applications where SDS concentrations are in the range of 0.05 to 1.0% SDS. In summary, we have developed a precise spectrophotometric method for determining the concentration of SDS in aqueous solutions. The method is easy to implement, accurate, and more robust than its predecessor in either a macrotiter format (cuvette at A453) or a microtiter format (cuvette at A450 or A453). This assay will be useful in any laboratory that has simple spectrophotometric absorbance capabilities and needs to measure SDS.
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Appendix A. Supplementary data
174 175
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.06.013.
176
References
152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
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[1] S. Trimpin, B. Brizzard, Analysis of insoluble proteins, Biotechniques 46 (2009) 409–419. [2] L.M. Hjelmeland, P.D. Murray, Solubilization of native membrane proteins, Methods Enzymol. 182 (1990) 253–264. [3] T. Arnold, D. Linke, The use of detergents to purify membrane proteins, Curr. Protoc. Protein Sci. (2001). unit 4.8.1–4.8.30. [4] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [5] S.U. Mehta, K.R. Rupprecht, J.C. Hunt, D.E. Kramer, B.J. McRae, R.G. Allen, G.J. Dawson, S.G. Devare, Prevalence of antibodies to the core protein P17, a serological marker during HIV-1 infection, AIDS Res. Hum. Retroviruses 6 (1990) 443–454.
3
[6] P.S. Brookes, A. Pinner, A. Ramachandran, L. Coward, S. Barnes, H. Kim, V.M. Darley-Usmar, High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes, Proteomics 2 (2002) 969–977. [7] T. Rae, R. Bonn, E. Lang, S. Stamenova, G. Burgos, K. Rupprecht, J. Fishpaugh, Utility of a direct dual-mode development analysis on blotted protein mixtures, Anal. Biochem. 415 (2011) 116–125. [8] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc. Natl. Acad. Sci. U.S.A. 76 (1979) 4350–4354. [9] J.-L. Rigaud, G. Mosser, J.-J. Lacapere, A. Olofsson, D. Levy, J.-L. Ranck, BioBeads: an efficient strategy for two-dimensional crystallization of membrane proteins, J. Struct. Biol. 118 (1997) 226–235. [10] K. Weber, M. Osborn, Proteins and sodium dodecyl sulfate: molecular weight determination on polyacrylamide gels and related procedures, in: H. Neurath, R.L. Hill (Eds.), The Proteins, Academic Press, New York, 1975, pp. 179–199. [11] H. Suzuki, T. Terada, Removal of dodecyl sulfate from protein solution, Anal. Biochem. 172 (1988) 259–263. [12] U. Carraro, D. Doria, C. Rizzi, M. Sandri, A new two-step precipitation method removes free-SDS and thiol reagents from diluted solutions, and then allows recovery and quantitation of proteins, Biochem. Biophys. Res. Commun. 200 (1994) 916–924. [13] J.R. Wis´niewski, D.F. Zielinska, M. Mann, Comparison of ultrafiltration units for proteomic and N-glycoproteomic analysis by the filter-aided sample preparation method, Anal. Biochem. 410 (2011) 307–309. [14] J.L. Fox, S.E. Stevens Jr., C.P. Taylor Jr., L.L. Poulsen, SDS removal from protein by polystyrene beads, Anal. Biochem. 87 (1978) 253–256. [15] M. Arand, T. Friedberg, F. Oesch, Colorimetric quantitation of trace amounts of sodium lauryl sulfate in the presence of nucleic acids and proteins, Anal. Biochem. 207 (1992) 73–75. [16] R. Bhattacharya, D. Mukherjee, D. Bhattacharyya, Quantification of proteinbound sodium dodecyl sulfate by rhodamine B: a method for identification of kinetically stable proteins, Anal. Biochem. 417 (2011) 17–24. [17] R. Campbell, M.A. Winkler, H. Wu, Quantification of sodium dodecyl sulfate in microliter biochemical samples by gas chromatography, Anal. Biochem. 335 (2004) 98–102. [18] J.E. Newbery, The variation of the critical micelle concentration of sodium dodecyl sulphate with ionic strength monitored by selective-ion membrane electrodes, Colloid Polym. Sci. 257 (1979) 773–775. [19] R. Roosloot, P. Schoen, A colorimetric assay for determination of residual detergent levels in reconstituted membrane protein preparations, Anal. Biochem. 413 (2011) 72–74. [20] F. Rusconi, É. Valton, R. Nguyen, E. Dufourc, Quantification of sodium dodecyl sulfate in microliter-volume biochemical samples by visible light spectroscopy, Anal. Biochem. 295 (2001) 31–37. [21] M. Mokus, U. Kragh-Hansen, P. Letellier, M. le Maire, J.V. Møller, Construction and use of a detergent-sensitive electrode to measure dodecyl sulfate activity and binding, Anal. Biochem. 264 (1998) 34–40. [22] S. Bullock, Fast quantification of sodium dodecyl sulfate in detergent by HPLC with ELSD, Application note SI-01218, Varian, 2008.
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Please cite this article in press as: K.R. Rupprecht et al., A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.06.013
No. of Pages 3, Model 5G
13 July 2015 Analytical Biochemistry xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7
A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration
5 8 9 10
11 1 4 3 2 14 15 16 17 18 19 20 21 22 23
Kevin R. Rupprecht ⇑, Ewa Z. Lang, Svetoslava D. Gregory, Janet M. Bergsma, Tracey D. Rae, Jeffrey R. Fishpaugh Diagnostics Analytical Chemistry Research, Abbott Laboratories, Abbott Park, IL 60049, USA
a r t i c l e
i n f o
Article history: Received 9 February 2015 Received in revised form 15 May 2015 Accepted 6 June 2015 Available online xxxx Keywords: Sodium dodecyl sulfate (SDS) Concentration Quantitative analysis
a b s t r a c t Sodium dodecyl sulfate (SDS) is used to denature and solubilize proteins, especially membrane and other hydrophobic proteins. A quantitative method to determine the concentration of SDS using the dye Stains-All is known. However, this method lacks the accuracy and reproducibility necessary for use with protein solutions where SDS concentration is a critical factor, so we modified this method after examining multiple parameters (solvent, pH, buffers, and light exposure). The improved method is simple to implement, robust, accurate, and (most important) precise. Ó 2015 Elsevier Inc. All rights reserved.
25 26 27 28 29 30 31 32
33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57
Sodium dodecyl sulfate (SDS)1 is a widely used detergent in molecular biology and biochemistry, including protein purification [1–3] and analysis [4–8]. Its utility is offset somewhat because it is difficult to remove from the protein once added to a solution [9– 14]. Quantification of SDS concentration in protein solutions is of interest, yet few quantitative assays are both practical and precise. Several methods are known, but none has been widely adopted [15–22]. One method, although both accurate and precise, relies on the separation of SDS into an organic phase [15]. This method is time-consuming and requires extensive extraction with chloroform. The most attractive spectroscopic method uses the interaction between SDS and a dye (Stains-All), where the method is easy to perform and has a broad dynamic range of 0.35 to 70 mM (0.01–2.0% SDS) [20]. An additional advantage is that the authors also demonstrated that a large number of potential sample matrix components, including protein and nucleic acids that are known to be stained by Stains-All, had no effect on the absorption at 438 nm, the recommended wavelength for analysis. Unfortunately, we obtained coefficients of variation (CVs) of 10 to 30% using this method and needed much better precision for our use. This article describes a modification of the Stains-All method with improved precision, mitigation of dye solubility issues, reduced background, and reduced sample buffer matrix effects. The method has a dynamic range of 0.001 to 0.01% ⇑ Corresponding author. Fax: +1 224 668 3271. E-mail address: [email protected] (K.R. Rupprecht). 1 Abbreviations used: SDS, sodium dodecyl sulfate; CV, coefficient of variation; LOD, limit of detection; LOQ, limit of quantitation; DMF, N,N-dimethylformamide; AU, absorption units.
SDS, making it usable with a single dilution for protein solutions with SDS concentrations of 0.05 to 1.0%. Precision is generally high (1–5%CV), with defined limits of detection (LOD) and quantitation (LOQ) of SDS. A detailed examination of the protocol [20] revealed several areas for potential improvement. The dye stock solution was problematic because significant amounts of undissolved dye were observed in multiple preparations of stock solution. The solution was filtered; however, this led to an unknown final concentration of the dye and differing adsorption peak intensities, and the dye continued to precipitate over time at 2 to 8 °C. An alternative stock solution using N,N-dimethylformamide (DMF) dissolved the dye completely, consistently, and at dye concentrations greater than 2 mg/ml. The subsequent working solution originally included the solvent formamide, but we found that by using the above DMF stock solution, a working solution could be prepared by simple dilution with water. Effects of buffers on the dye spectra were also investigated. A buffer of 10 mM sodium phosphate and 15 mM NaCl at pH 7.2 was eventually chosen because it gave the best combination of absorbance and separation of wavelengths between SDS-bound Stains-All and unbound dye (see the online Supplementary material for additional discussion of the effects of various buffers). In addition, the wavelength previously used for quantification, 438 nm [20], was found to be on the side of the absorbance peak rather than at the apex (see Fig. 1), and the kmax varied from 445 to 460 nm. The steepness of the peak slope combined with the varying kmax led to poorer precision in our experience than reported by the original assay authors [20] and,
http://dx.doi.org/10.1016/j.ab.2015.06.013 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.R. Rupprecht et al., A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.06.013
58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85
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Notes & Tips / Anal. Biochem. xxx (2015) xxx–xxx
Fig.1. Spectra of SDS bound to Stains-All. Absorbance spectra of Stains-All with bound SDS solutions at 0, 0.001, 0.0025, 0.005, 0.0075, and 0.01% SDS are shown, where kmax (453 nm) is indicated as well as the wavelength previously used for measuring SDS concentration (438 nm) [20]. The spectra indicate that 438 nm is not optimal for precision. The data for these solutions were plotted to demonstrate assay linearity (R = 0.998).
86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125
subsequently, to higher variation in SDS concentration. We used the A453 for quantitation to improve precision in our assay, and CVs decreased from P30 to 65.4%. Dyes can be photosensitive, and for our initial solutions there was an overall loss of more than 50% absorbance within 1 day, and the sensitivity to light is exacerbated with high concentrations of protein (100 lg/ml) in the final assay solution. The previous work [20] demonstrated that protein at a low concentration (4.33 lg/ml) in the final assay solution did not affect the absorbance of the SDS–Stains-All complex. Our sample preparation dilution yielded protein concentrations of less than 2 lg/ml in the final assay solution, and light-sensitive signal loss is minimized to less than 5%. The detailed protocol can be found in the Supplementary material. Our above modifications of the previous method [20] were developed using a cuvette assay measured with a spectrophotometer (macro format), and we adapted these modifications to a microtiter format. Depending on the microtiter system used, instrumentation limits may require a shift in the wavelength to 450 nm when using filter-based instruments, such as the ELx 808 Ultra Microplate Reader, that use a filter with a bandwidth of 440 to 460 nm. The data below were obtained using the ELx 808 Ultra Microplate Reader. Both the cuvette and the microtiter assays were designed to provide analysis of samples in the same concentration range, with similar assay performance characteristics. Assay performance was examined by linear fit, precision, reproducibility, LOD, and LOQ using the standards that ranged from 0.001 to 0.01% with water as a blank. The concentration of SDS was found to be highly linear over this range in both the cuvette and microtiter assays (A450 and A453). Standards were run in triplicate, with R values of 0.989 to 0.999 for the cuvette assay and 0.998 to 0.999 for the microtiter assay. Percentage CVs were determined for triplicate analyses to assess within-assay precision and were found to be lower for the microtiter assay (see Table 1; see also Table S2 in the Supplementary material). The lowest calibrator (0.001%) had CVs of 5.4% (cuvette) and 4.7% (microtiter), whereas the highest calibrator (0.01%) had CVs of 2.5 and 1.5%, respectively. Two types of control samples were used to aid in assessing performance; one set had concentrations of SDS in water (0.0033 and 0.0066%), and a second consisted of two protein solutions containing SDS (0.15 and 0.5%). The proteins used were proprietary
Table 1 Comparison of assay precision and reproducibility between the cuvette assay measured at 453 nm and the microtiter assay measured at 450 nm. Control
n
Cuvette average value
Cuvette average %CV
Microtiter average value
Microtiter average %CV
Control A (0.0033% SDS) Control B (0.0067% SDS) Sample control A Sample control B Linearity (R)
8
0.0035
3.46
0.0033
2.05
8
0.0074
2.98
0.0065
1.31
8 8 8
0.1491 0.4993 0.9964
2.46 3.68 0.69
0.1635 0.4857 0.9987
1.48 1.40 0.08
recombinant proteins having molecular masses of approximately 46 and 23 kDa. The microtiter assay again gave tighter CVs than the cuvette assay format, most likely due to the improved precision in pipetting the components. In the cuvette assay, the average CVs for the two aqueous SDS controls were 3.5 and 3.0%, with 2.5 and 3.7% for the two protein–SDS controls. In the microtiter assay, we observed average CVs of 2.1 and 1.3% for the aqueous SDS controls, with 1.5 and 1.4% for the two protein–SDS controls. We also compared the filter-based reader with a monochromator-based system, the Synergy Mx. The two microtiter systems produced highly comparable results (see Table S3). Reproducibility was examined in-depth using the microtiter format with three different lots of Stains-All purchased over a 3-year time range to determine lot-to-lot precision and consistency of the SDS–Stains-All interaction. All of the lots examined gave good precision, with CVs ranging from 0.27 to 3.42% for the standard solutions. The A450 absorbance of the high controls analyzed using the three Stains-All lots varied from the mean of the three lots by 0.012 to 0.026 absorption units (AU) (1.162 AU mean) and 0.006 to 0.011 AU (0.6336 AU mean) for the low control lots. Final SDS concentrations for all samples and controls varied from each other by ±3.1% for all three dye lots tested. Operator-to-operator variability was examined and produced CVs of less than 5.3% for all samples, controls, and standards, with the highest CVs seen with the lowest standard. Between-operator comparisons showed CVs of less than 4.9% for all samples and
Please cite this article in press as: K.R. Rupprecht et al., A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.06.013
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controls. Finally, a series of assays was used to determine LODs and LOQs in both the cuvette and microtiter assays. LOD was taken as 3 noise, with noise determined as the greatest difference observed between 24 blank determinations, and 10 noise was used to determine LOQ. For the cuvette assay, an LOD of 0.0004% SDS and an LOQ of 0.0008% SDS was achieved, whereas the LOD and LOQ for the microtiter assay were 0.0006 and 0.0008% SDS, respectively. The higher LOD for the microtiter assay reflects the shorter AU range achievable in the microtiter reader due to the shorter path length relative to the spectrophotometer used in the cuvette assay. Overall, both assays produced superior performance to other published methods and can be used for most biological applications where SDS concentrations are in the range of 0.05 to 1.0% SDS. In summary, we have developed a precise spectrophotometric method for determining the concentration of SDS in aqueous solutions. The method is easy to implement, accurate, and more robust than its predecessor in either a macrotiter format (cuvette at A453) or a microtiter format (cuvette at A450 or A453). This assay will be useful in any laboratory that has simple spectrophotometric absorbance capabilities and needs to measure SDS.
173
Appendix A. Supplementary data
174 175
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.06.013.
176
References
152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171
177 178 179 180 181 182 183 184 185 186 187 188
[1] S. Trimpin, B. Brizzard, Analysis of insoluble proteins, Biotechniques 46 (2009) 409–419. [2] L.M. Hjelmeland, P.D. Murray, Solubilization of native membrane proteins, Methods Enzymol. 182 (1990) 253–264. [3] T. Arnold, D. Linke, The use of detergents to purify membrane proteins, Curr. Protoc. Protein Sci. (2001). unit 4.8.1–4.8.30. [4] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [5] S.U. Mehta, K.R. Rupprecht, J.C. Hunt, D.E. Kramer, B.J. McRae, R.G. Allen, G.J. Dawson, S.G. Devare, Prevalence of antibodies to the core protein P17, a serological marker during HIV-1 infection, AIDS Res. Hum. Retroviruses 6 (1990) 443–454.
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Please cite this article in press as: K.R. Rupprecht et al., A precise spectrophotometric method for measuring sodium dodecyl sulfate concentration, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.06.013