- Email: [email protected]
Sodium dodecyl sulfate polyacrylamide gel electrophoresis for direct quantitation of protein adsorption
Accepted Manuscript Notes & tips SDS-PAGE for Direct Quantitation of Protein Adsorption Man Chung Gilbert Lee, Kinny Sheung Yang Wu, Tam N.T. Nguyen, Bingyun Sun PII: DOI: Reference:
S0003-2697(14)00336-4 http://dx.doi.org/10.1016/j.ab.2014.07.031 YABIO 11823
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
25 May 2014 26 July 2014 28 July 2014
Please cite this article as: M.C.G. Lee, K.S.Y. Wu, T.N.T. Nguyen, B. Sun, SDS-PAGE for Direct Quantitation of Protein Adsorption, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.07.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
SDS-PAGE for Direct Quantitation of Protein Adsorption
Man Chung Gilbert Lee, Kinny Sheung Yang Wu, Tam N.T. Nguyen and Bingyun Sun*
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada
*To whom correspondence should be addressed: Department of Chemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada Tel.: 778-782-9097, Fax: 778-782-3765, E-mail: [email protected]
Short title: SDS-PAGE/DPA Quantitation of Protein Adsorption
Keywords: protein adsorption, sample loss, SDS-PAGE, SDS-PAGE/DPA, sensitive protein analysis, single-cell proteomics
1
2
ABSTRACT A simple method, SDS-PAGE coupled with Direct Protein Analysis (SDSPAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved, and this method is capable of measuring proteins adsorbed on sample vials at extremely low concentration (in pg/uL). We used this method to characterize the effect of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures, and its utility in developing preventative strategies of adsorption.
3
Proteins in solution can spontaneously adsorb to solid surfaces, and the adsorption event itself has raised many concerns in the fields of biology, medicine, material science and engineering [1]. For example, in sensitive protein analyses, adsorption is a major source of sample loss that has largely hindered researches carried out in single-cell proteomics [2]. The process of protein adsorption is complex and protein-dependent, based heavily on the protein size, structure, and hydrophobicity to name a few [3, 4]. The studies of adsorption, however, have been limited to a handful of model proteins, mostly blood and milk proteins, using special equipment, such as quartz crystal microbalance [5], ellipsometer [6], and neutron reflector [7]. Methods that do not rely on these instruments are likely to use certain labels such as fluorophores [8, 9] and radioisotopes [10-13] to enhance the detection sensitivity. These labels are known to modify the structure and, therefore, adsorptivity of intact proteins [12, 14]. A frequently used labelfree method --- “solution-depletion” method --- employs SDS-PAGE instead of expensive instruments to measure the changes of protein concentration before and after the contact of solid surfaces [8, 15]. Nevertheless, the method is limited to systems with large surface areas such as beads or small particles [12, 15, 16]. No simple and sensitive label-free methods are available to characterizing protein adsorption in regular sample vials. To extend the use of SDS-PAGE to regular vials with smaller and flatter surfaces than beads, we report here a method that couples SDS-PAGE with “rinse-stripping” based Direct Protein-Adsorption analysis (SDS-PAGE/DPA). Previously, “rinsestripping” method was impossible to be coupled to SDS-PAGE, due to the relatively large volume and low concentration of protein in the resolved stripping solution. We
4
overcome these difficulties by concentrating the obtained stripping solution into small volume. This simple change makes SDS-PAGE capable of measuring adsorption occurred in low pg/uL protein solution and regular sample vials. We demonstrate below the utility of this method in characterizing the effect of concentration, volume, and time on adsorption, and the competitive and sequential adsorption in a protein mixture. Bovine Serum Albumins (BSA) (Sigma, cat. no A9647) stock solution in phosphate-buffered saline (PBS) was diluted directly in 0.65-mL micro-centrifuge vials (VWR, 870ac03-290) to 200 uL working solution, if not further specified. All the proceeding incubations were carried out in vials with upright-position at room temperature and no agitation. After the defined duration, solutions were removed, and the vials were rinsed three times with PBS, first by pipetting and then by two times of vortexing. After the final wash, 200-uL 0.5 wt% SDS was introduced to these vials to strip the adsorbed protein molecules. All the wash and stripping solutions were collected and dried in Speedvec (Thermo Scientific) prior to SDS-PAGE (Bio-Rad) analysis. These major steps are summarized in Figure 1A, and all experiments in our studies have replicates. SDS-PAGE analyses were carried out with 15% polyacrylamide gel and silver staining following the standard protocols [17, 18]. Dried samples were first reconstituted in sample buffer, boiled for 10 minutes at 100oC, and then cooled to room temperature before being loaded to the gel. For absolute quantitation, a set of protein standards were applied, and the resolved gel was digitized by LiDE 110 scanner (Canon, Canada Inc.) and converted to intensity profiles using ImageJ (http://rsbweb.nih.gov/ij/). Quantitation was based on the total intensity of protein bands.
5
To verify the method, we characterized the adsorption from 1ug/uL BSA for 1 h incubation in three repeated experiments. The wash and stripping efficiency is summarized in Figures 1B & 1C. Figure 1B shows the SDS-PAGE image of BSA resolved from each wash (W) and stripping (T) solution as well as from the standard protein (Sd.). The bar chart in Figure 1C shows the average quantity of BSA in each step of the repeated experiments. The percentage of the obtained BSA molecules over the total protein quantity in the initial solution is provided on top of each bar in Figure 1C. The R2 value of the standard curve is more than 0.9, the error bars mark the standard deviation of the measurement, and the coefficient of variance (CV) of all the BSA values are less than 6%, suggesting a good accuracy and reproducibility of the method, which agrees with the existing knowledge of SDS-PAGE [19]. The exponential drop of BSA quantity in the three consecutive washes and the marginal residual BSA (0.03%) in the final wash in Figure 1C indicate the wash scheme we applied is adequate. We also used additional basic and organic solvent to strip the vials after SDS, and only recovered less than 0.5% of the SDS-stripped BSA molecules as detailed in the supplementary materials and Figure S1. Therefore, our stripping condition is sufficient to recover the majority of the adsorbed molecules. The measured adsorptivity of BSA on the polypropylene surface of our microcentrifuge vials is 0.3 ug/cm2, which matches the reported values of BSA adsorbed to hydrophobic plastic materials [3, 4, 11, 14]. We characterized the detection sensitivity of SDS-PAGE/DPA by varying the concentrations of BSA for 1-h incubation as shown in Figures 2A & 2B. In these Figures, the least detectable BSA adsorption is from 100 pg/uL protein solution. By extending the silver staining time, we can quantify adsorption from as low as 4 pg/uL protein solutions,
6
in which complete loss of protein can be detected (supplementary Figure S2). We further characterized the time effect of adsorption by varying the incubation duration of 1-ng/uL BSA. Figures 2C & 2D summarize the results, in which the surface occupancy was observed in the first 10 seconds of the solution contact. Such a quick event suggests that adsorption-caused protein loss can occur in any protein handling procedures without prolonged storage. Therefore, it is crucial to study how various proteins adsorb to sample vials in any studies concerning low concentration of proteins, especially in those with multiple steps, such as the single-cell proteomics. Through the characterization of the volume effect as detailed in the supplementary material, we further verified that the smallest surface can be measured by our method is 0.57 cm2 occupied by 20 uL of BSA at 200 ng/uL concentration (supplementary Figure S3). To compare the improvements of this method over the existing “solution-depletion” method, we performed the solutiondepletion analysis on BSA incubated in vials without beads or particles. Within the detection limit of SDS-PAGE/DPA, no adsorption was detectable via the “solutiondepletion” method (supplementary Figure S4). Finally we demonstrated that SDS-PAGE/DPA was possible to study differential adsorption when competitive proteins are present such as those in a protein mixture. The usage of SDS-PAGE in characterizing competitive protein adsorption has been demonstrated by “solution-depletion” method (15); our experiments here were to test whether the DPA approach would work equally well. We used equal concentration of BSA to compete with 200-ng/uL
equine skeletal-muscle myoglobin (MB) (Sigma, cat. no. M0630) for 1-h adsorption, and compared the results with the adsorption of these proteins alone. Figures 2E and 2F summarize the results, labeled as (BSA/MB). The presence of BSA significantly decreased the surface adsorption of MB (from 0.5 ug to 0.04 ug, > 10 fold), whereas MB 7
did not affect BSA adsorption much (from 0.5 ug to 0.31ug, < 1 fold); the adsorptivity of BSA and MB in equal concentration mixture is different, with BSA (0.3 ug) adsorbing about 8 fold more than that of MB (0.04 ug). Such a large difference in adsorption can significantly impact the accuracy of quantitative analysis. Because of the superior surface affinity of BSA, we furthered our experiment by coating the sample vials with 200 ng/uL BSA for 1 h and then applying equal concentration of MB for another 1 h after rinsing the free BSA with PBS. The sequential results (labeled as BSA-MB) are also included in Figures 2E &2F, and are similar to the competitive results (BSA/MB). Pre-coating the vials with BSA in our experiments decreased MB adsorption about 6-fold. Even though we demonstrated here the analysis of a two-protein mixture, our method can readily detect differential adsorption taken place in complex protein mixtures, and facilitate the development of sample-specific preventative strategies. The separation power of SDSPAGE permits the mixing of BSA and MB standards during analysis as shown in Figure 2E, which is more convenient than other methods in which each standard requires a separate measurement. In cases where similar molecular-weight proteins are present, orthogonal separations or analyses, such as 2D–PAGE or MS can be used to increase separation efficiency as demonstrated in many proteomics dealing with complex biological samples. In short, we introduce here the SDS-PAGE/DPA method that is sensitive to quantify protein molecules adsorbed on smaller and flatter surfaces, to which the solution-depletion method cannot apply. The method has wide applications including competitive adsorption in protein mixtures and the sequential adsorption that can be used to prevent the loss of the target proteins. No special equipment is needed in this method;
8
therefore many labs are able to carry out these analyses. We hope this simple and easy method can help enrich our understanding of protein adsorption from information collected by different researchers, and also help develop strategies to improve the sensitivity and accuracy of studies concerning proteins with low concentrations or samples with limited materials such as those in single-cell proteomics.
ACKNOWLEDGMENT This research is supported by Simon Fraser University and Stem Cell Network in Canada.
9
REFERENCES [1] Sadana A. Protein Adsorption and Inactivation on Surfaces. Influence of Heterogeneities. Chem Rev. 1992;92:1799-818. [2] Wang D, Bodovitz S. Single cell analysis: the new frontier in 'omics'. Trends Biotechnol. 2010;28:281-90. [3] Nakanishi K, Sakiyama T, Imamura K. On the Adsorption of Proteins on Solid Surfaces, a Common but Very Complicated Phenomenon. Journal of Bioscience and Bioengineering. 2001;91:233-44. [4] Rabe M, Verdes D, Seeger S. Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interface Sci. 2011;162:87-106. [5] Reimhult K, Petersson K, Krozer A. QCM-D Analysis of the Performance of Blocking Agents on Gold and Polystyrene Surfaces. Langmuir. 2008;24:8695-700. [6] Benesch J, Askendal A, Tengvall P. Quantification of adsorbed human serum albumin at solid interfaces: a comparison between radioimmunoassay (RIA) and simple null ellipsometry. Colloids and Surfaces B: Biointerfaces. 2000;18:71-81. [7] Brouette N, Fragneto G, Cousin F, Moulin M, Haertlein M, Sferrazza M. A neutron reflection study of adsorbed deuterated myoglobin layers on hydrophobic surfaces. J Colloid Interface Sci. 2013;390:114-20. [8] Varmette E, Strony B, Haines D, Redkar R. An assay measuremernt of protein adsorption to glass vials. Journal of Pharmaceutical Science and Technology. 2010;64:305. [9] Verzola B, Gelfi C, Righetti PG. Protein adsorption to the bare silica wall in capillary electrophoresis Quantitative study on the chemical composition of the background
10
electrolyte for minimising the phenomenon. Journal of Chromatography A. 2000;868:8599. [10] Dupont-Gillain CC, Fauroux CMJ, Gardner DCJ, Leggett GJ. Use of AFM to probe the adsorption strength and time-dependent changes of albumin on self-assembled monolayers. Journal of Biomedical Materials Research Part A. 2003;67A:548-58. [11] Holmberg M, Hou X. Competitive protein adsorption of albumin and immunoglobulin G from human serum onto polymer surfaces. Langmuir. 2009;26:93842. [12] Leibner ES, Barnthip N, Chen W, Baumrucker CR, Badding JV, Pishko M, et al. Superhydrophobic effect on the adsorption of human serum albumin. Acta Biomater. 2009;5:1389-98. [13] Suelter CH, Deluca M. How to Prevent Losses of Protein by Adsorption to Glass and Plastic. Analytical Biochemistry. 1983;135:112-9. [14] Vogler EA. Protein adsorption in three dimensions. Biomaterials. 2012;33:1201-37. [15] Noh H, Barnthip N, Parhi P, Vogler EA. Electrophoretic implementation of the solution-depletion method for measuring protein adsorption, adsorption kinetics, and adsorption competition among multiple proteins in solution. Methods in molecular biology. 2013;1025:157-66. [16] Hu G, Heitmann JJA, Rojas OJ, Pawlak JJ, Argyopoulos DS. Monitoring Cellulase Protein Adsorption and Recovery Using SDS-PAGE. Ind Eng Chem Res. 2010;49:83338. [17] Gromova I, Celis JE. Protein detection in gels by silver staining: A procedure compatible with mass-spectrometry. In: Celis JE, Carter N, Hunter T, Simons K, Small
11
JV, Shotton D, editors. Cell Biology: A Laboratory Handbook. 3 ed. San Diego: Elsevier. Academic Press; 2006. p. 421-9. [18] Switzer III RC, Merril CR, Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Analytical Biochemistry. 1979;98:231–7. [19] Noh H, Vogler EA. Volumetric interpretation of protein adsorption: Partition coefficients, interphase volumes, and free energies of adsorption to hydrophobic surfaces. Biomaterials. 2006;27:5780-93.
12
FIGURE CAPTIONS
Figure 1. Illustration of the experimental procedure and the characterization of SDSPAGE/DPA. (A) Flow chart of SDS-PAGE/DPA. (B) SDS-PAGE image of BSA molecules obtained in three washes (W-1, W-2, and W-3) and the final stripping solution (T) in three repeated experiments. Sd. stands the BSA standard. (C) Average quantity of the adsorbed BSA molecules in all the solutions and the correspondent standard curve with the R2 value. The percentage of the resolved BSA relative to the total initial is listed on top of each bar.
Figure 2. Applications of SDS-PAGE/DPA. (A) SDS-PAGE image of the adsorbed BSA in six different concentrations with replicates (condition: 200-uL BSA and 1-h incubation). (B) Bar chart of BSA quantity in (A). (C) SDS-PAGE image of adsorbed BSA in different incubation time with replicates (condition: 200 uL of 1 ng/uL BSA). (D) Bar chart of BSA quantity in (C). (E) SDS-PAGE image of the adsorption from pure BSA and MB solutions, as well as from competitive (BSA/MB) and sequential (BSAMB) adsorption with replicates. (F) Quantity of the adsorbed BSA and MB in (E).
13
Figure 1
14
Figure 2
15
S0003-2697(14)00336-4 http://dx.doi.org/10.1016/j.ab.2014.07.031 YABIO 11823
To appear in:
Analytical Biochemistry
Received Date: Revised Date: Accepted Date:
25 May 2014 26 July 2014 28 July 2014
Please cite this article as: M.C.G. Lee, K.S.Y. Wu, T.N.T. Nguyen, B. Sun, SDS-PAGE for Direct Quantitation of Protein Adsorption, Analytical Biochemistry (2014), doi: http://dx.doi.org/10.1016/j.ab.2014.07.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
SDS-PAGE for Direct Quantitation of Protein Adsorption
Man Chung Gilbert Lee, Kinny Sheung Yang Wu, Tam N.T. Nguyen and Bingyun Sun*
Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada
*To whom correspondence should be addressed: Department of Chemistry, Simon Fraser University, Burnaby, BC V5A1S6, Canada Tel.: 778-782-9097, Fax: 778-782-3765, E-mail: [email protected]
Short title: SDS-PAGE/DPA Quantitation of Protein Adsorption
Keywords: protein adsorption, sample loss, SDS-PAGE, SDS-PAGE/DPA, sensitive protein analysis, single-cell proteomics
1
2
ABSTRACT A simple method, SDS-PAGE coupled with Direct Protein Analysis (SDSPAGE/DPA), is presented here for the quantitation of adsorption-caused protein loss. No complicated steps and expensive equipment are involved, and this method is capable of measuring proteins adsorbed on sample vials at extremely low concentration (in pg/uL). We used this method to characterize the effect of concentration, time, and volume on adsorption. We also applied this method to discover differential sample loss in protein mixtures, and its utility in developing preventative strategies of adsorption.
3
Proteins in solution can spontaneously adsorb to solid surfaces, and the adsorption event itself has raised many concerns in the fields of biology, medicine, material science and engineering [1]. For example, in sensitive protein analyses, adsorption is a major source of sample loss that has largely hindered researches carried out in single-cell proteomics [2]. The process of protein adsorption is complex and protein-dependent, based heavily on the protein size, structure, and hydrophobicity to name a few [3, 4]. The studies of adsorption, however, have been limited to a handful of model proteins, mostly blood and milk proteins, using special equipment, such as quartz crystal microbalance [5], ellipsometer [6], and neutron reflector [7]. Methods that do not rely on these instruments are likely to use certain labels such as fluorophores [8, 9] and radioisotopes [10-13] to enhance the detection sensitivity. These labels are known to modify the structure and, therefore, adsorptivity of intact proteins [12, 14]. A frequently used labelfree method --- “solution-depletion” method --- employs SDS-PAGE instead of expensive instruments to measure the changes of protein concentration before and after the contact of solid surfaces [8, 15]. Nevertheless, the method is limited to systems with large surface areas such as beads or small particles [12, 15, 16]. No simple and sensitive label-free methods are available to characterizing protein adsorption in regular sample vials. To extend the use of SDS-PAGE to regular vials with smaller and flatter surfaces than beads, we report here a method that couples SDS-PAGE with “rinse-stripping” based Direct Protein-Adsorption analysis (SDS-PAGE/DPA). Previously, “rinsestripping” method was impossible to be coupled to SDS-PAGE, due to the relatively large volume and low concentration of protein in the resolved stripping solution. We
4
overcome these difficulties by concentrating the obtained stripping solution into small volume. This simple change makes SDS-PAGE capable of measuring adsorption occurred in low pg/uL protein solution and regular sample vials. We demonstrate below the utility of this method in characterizing the effect of concentration, volume, and time on adsorption, and the competitive and sequential adsorption in a protein mixture. Bovine Serum Albumins (BSA) (Sigma, cat. no A9647) stock solution in phosphate-buffered saline (PBS) was diluted directly in 0.65-mL micro-centrifuge vials (VWR, 870ac03-290) to 200 uL working solution, if not further specified. All the proceeding incubations were carried out in vials with upright-position at room temperature and no agitation. After the defined duration, solutions were removed, and the vials were rinsed three times with PBS, first by pipetting and then by two times of vortexing. After the final wash, 200-uL 0.5 wt% SDS was introduced to these vials to strip the adsorbed protein molecules. All the wash and stripping solutions were collected and dried in Speedvec (Thermo Scientific) prior to SDS-PAGE (Bio-Rad) analysis. These major steps are summarized in Figure 1A, and all experiments in our studies have replicates. SDS-PAGE analyses were carried out with 15% polyacrylamide gel and silver staining following the standard protocols [17, 18]. Dried samples were first reconstituted in sample buffer, boiled for 10 minutes at 100oC, and then cooled to room temperature before being loaded to the gel. For absolute quantitation, a set of protein standards were applied, and the resolved gel was digitized by LiDE 110 scanner (Canon, Canada Inc.) and converted to intensity profiles using ImageJ (http://rsbweb.nih.gov/ij/). Quantitation was based on the total intensity of protein bands.
5
To verify the method, we characterized the adsorption from 1ug/uL BSA for 1 h incubation in three repeated experiments. The wash and stripping efficiency is summarized in Figures 1B & 1C. Figure 1B shows the SDS-PAGE image of BSA resolved from each wash (W) and stripping (T) solution as well as from the standard protein (Sd.). The bar chart in Figure 1C shows the average quantity of BSA in each step of the repeated experiments. The percentage of the obtained BSA molecules over the total protein quantity in the initial solution is provided on top of each bar in Figure 1C. The R2 value of the standard curve is more than 0.9, the error bars mark the standard deviation of the measurement, and the coefficient of variance (CV) of all the BSA values are less than 6%, suggesting a good accuracy and reproducibility of the method, which agrees with the existing knowledge of SDS-PAGE [19]. The exponential drop of BSA quantity in the three consecutive washes and the marginal residual BSA (0.03%) in the final wash in Figure 1C indicate the wash scheme we applied is adequate. We also used additional basic and organic solvent to strip the vials after SDS, and only recovered less than 0.5% of the SDS-stripped BSA molecules as detailed in the supplementary materials and Figure S1. Therefore, our stripping condition is sufficient to recover the majority of the adsorbed molecules. The measured adsorptivity of BSA on the polypropylene surface of our microcentrifuge vials is 0.3 ug/cm2, which matches the reported values of BSA adsorbed to hydrophobic plastic materials [3, 4, 11, 14]. We characterized the detection sensitivity of SDS-PAGE/DPA by varying the concentrations of BSA for 1-h incubation as shown in Figures 2A & 2B. In these Figures, the least detectable BSA adsorption is from 100 pg/uL protein solution. By extending the silver staining time, we can quantify adsorption from as low as 4 pg/uL protein solutions,
6
in which complete loss of protein can be detected (supplementary Figure S2). We further characterized the time effect of adsorption by varying the incubation duration of 1-ng/uL BSA. Figures 2C & 2D summarize the results, in which the surface occupancy was observed in the first 10 seconds of the solution contact. Such a quick event suggests that adsorption-caused protein loss can occur in any protein handling procedures without prolonged storage. Therefore, it is crucial to study how various proteins adsorb to sample vials in any studies concerning low concentration of proteins, especially in those with multiple steps, such as the single-cell proteomics. Through the characterization of the volume effect as detailed in the supplementary material, we further verified that the smallest surface can be measured by our method is 0.57 cm2 occupied by 20 uL of BSA at 200 ng/uL concentration (supplementary Figure S3). To compare the improvements of this method over the existing “solution-depletion” method, we performed the solutiondepletion analysis on BSA incubated in vials without beads or particles. Within the detection limit of SDS-PAGE/DPA, no adsorption was detectable via the “solutiondepletion” method (supplementary Figure S4). Finally we demonstrated that SDS-PAGE/DPA was possible to study differential adsorption when competitive proteins are present such as those in a protein mixture. The usage of SDS-PAGE in characterizing competitive protein adsorption has been demonstrated by “solution-depletion” method (15); our experiments here were to test whether the DPA approach would work equally well. We used equal concentration of BSA to compete with 200-ng/uL
equine skeletal-muscle myoglobin (MB) (Sigma, cat. no. M0630) for 1-h adsorption, and compared the results with the adsorption of these proteins alone. Figures 2E and 2F summarize the results, labeled as (BSA/MB). The presence of BSA significantly decreased the surface adsorption of MB (from 0.5 ug to 0.04 ug, > 10 fold), whereas MB 7
did not affect BSA adsorption much (from 0.5 ug to 0.31ug, < 1 fold); the adsorptivity of BSA and MB in equal concentration mixture is different, with BSA (0.3 ug) adsorbing about 8 fold more than that of MB (0.04 ug). Such a large difference in adsorption can significantly impact the accuracy of quantitative analysis. Because of the superior surface affinity of BSA, we furthered our experiment by coating the sample vials with 200 ng/uL BSA for 1 h and then applying equal concentration of MB for another 1 h after rinsing the free BSA with PBS. The sequential results (labeled as BSA-MB) are also included in Figures 2E &2F, and are similar to the competitive results (BSA/MB). Pre-coating the vials with BSA in our experiments decreased MB adsorption about 6-fold. Even though we demonstrated here the analysis of a two-protein mixture, our method can readily detect differential adsorption taken place in complex protein mixtures, and facilitate the development of sample-specific preventative strategies. The separation power of SDSPAGE permits the mixing of BSA and MB standards during analysis as shown in Figure 2E, which is more convenient than other methods in which each standard requires a separate measurement. In cases where similar molecular-weight proteins are present, orthogonal separations or analyses, such as 2D–PAGE or MS can be used to increase separation efficiency as demonstrated in many proteomics dealing with complex biological samples. In short, we introduce here the SDS-PAGE/DPA method that is sensitive to quantify protein molecules adsorbed on smaller and flatter surfaces, to which the solution-depletion method cannot apply. The method has wide applications including competitive adsorption in protein mixtures and the sequential adsorption that can be used to prevent the loss of the target proteins. No special equipment is needed in this method;
8
therefore many labs are able to carry out these analyses. We hope this simple and easy method can help enrich our understanding of protein adsorption from information collected by different researchers, and also help develop strategies to improve the sensitivity and accuracy of studies concerning proteins with low concentrations or samples with limited materials such as those in single-cell proteomics.
ACKNOWLEDGMENT This research is supported by Simon Fraser University and Stem Cell Network in Canada.
9
REFERENCES [1] Sadana A. Protein Adsorption and Inactivation on Surfaces. Influence of Heterogeneities. Chem Rev. 1992;92:1799-818. [2] Wang D, Bodovitz S. Single cell analysis: the new frontier in 'omics'. Trends Biotechnol. 2010;28:281-90. [3] Nakanishi K, Sakiyama T, Imamura K. On the Adsorption of Proteins on Solid Surfaces, a Common but Very Complicated Phenomenon. Journal of Bioscience and Bioengineering. 2001;91:233-44. [4] Rabe M, Verdes D, Seeger S. Understanding protein adsorption phenomena at solid surfaces. Adv Colloid Interface Sci. 2011;162:87-106. [5] Reimhult K, Petersson K, Krozer A. QCM-D Analysis of the Performance of Blocking Agents on Gold and Polystyrene Surfaces. Langmuir. 2008;24:8695-700. [6] Benesch J, Askendal A, Tengvall P. Quantification of adsorbed human serum albumin at solid interfaces: a comparison between radioimmunoassay (RIA) and simple null ellipsometry. Colloids and Surfaces B: Biointerfaces. 2000;18:71-81. [7] Brouette N, Fragneto G, Cousin F, Moulin M, Haertlein M, Sferrazza M. A neutron reflection study of adsorbed deuterated myoglobin layers on hydrophobic surfaces. J Colloid Interface Sci. 2013;390:114-20. [8] Varmette E, Strony B, Haines D, Redkar R. An assay measuremernt of protein adsorption to glass vials. Journal of Pharmaceutical Science and Technology. 2010;64:305. [9] Verzola B, Gelfi C, Righetti PG. Protein adsorption to the bare silica wall in capillary electrophoresis Quantitative study on the chemical composition of the background
10
electrolyte for minimising the phenomenon. Journal of Chromatography A. 2000;868:8599. [10] Dupont-Gillain CC, Fauroux CMJ, Gardner DCJ, Leggett GJ. Use of AFM to probe the adsorption strength and time-dependent changes of albumin on self-assembled monolayers. Journal of Biomedical Materials Research Part A. 2003;67A:548-58. [11] Holmberg M, Hou X. Competitive protein adsorption of albumin and immunoglobulin G from human serum onto polymer surfaces. Langmuir. 2009;26:93842. [12] Leibner ES, Barnthip N, Chen W, Baumrucker CR, Badding JV, Pishko M, et al. Superhydrophobic effect on the adsorption of human serum albumin. Acta Biomater. 2009;5:1389-98. [13] Suelter CH, Deluca M. How to Prevent Losses of Protein by Adsorption to Glass and Plastic. Analytical Biochemistry. 1983;135:112-9. [14] Vogler EA. Protein adsorption in three dimensions. Biomaterials. 2012;33:1201-37. [15] Noh H, Barnthip N, Parhi P, Vogler EA. Electrophoretic implementation of the solution-depletion method for measuring protein adsorption, adsorption kinetics, and adsorption competition among multiple proteins in solution. Methods in molecular biology. 2013;1025:157-66. [16] Hu G, Heitmann JJA, Rojas OJ, Pawlak JJ, Argyopoulos DS. Monitoring Cellulase Protein Adsorption and Recovery Using SDS-PAGE. Ind Eng Chem Res. 2010;49:83338. [17] Gromova I, Celis JE. Protein detection in gels by silver staining: A procedure compatible with mass-spectrometry. In: Celis JE, Carter N, Hunter T, Simons K, Small
11
JV, Shotton D, editors. Cell Biology: A Laboratory Handbook. 3 ed. San Diego: Elsevier. Academic Press; 2006. p. 421-9. [18] Switzer III RC, Merril CR, Shifrin S. A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Analytical Biochemistry. 1979;98:231–7. [19] Noh H, Vogler EA. Volumetric interpretation of protein adsorption: Partition coefficients, interphase volumes, and free energies of adsorption to hydrophobic surfaces. Biomaterials. 2006;27:5780-93.
12
FIGURE CAPTIONS
Figure 1. Illustration of the experimental procedure and the characterization of SDSPAGE/DPA. (A) Flow chart of SDS-PAGE/DPA. (B) SDS-PAGE image of BSA molecules obtained in three washes (W-1, W-2, and W-3) and the final stripping solution (T) in three repeated experiments. Sd. stands the BSA standard. (C) Average quantity of the adsorbed BSA molecules in all the solutions and the correspondent standard curve with the R2 value. The percentage of the resolved BSA relative to the total initial is listed on top of each bar.
Figure 2. Applications of SDS-PAGE/DPA. (A) SDS-PAGE image of the adsorbed BSA in six different concentrations with replicates (condition: 200-uL BSA and 1-h incubation). (B) Bar chart of BSA quantity in (A). (C) SDS-PAGE image of adsorbed BSA in different incubation time with replicates (condition: 200 uL of 1 ng/uL BSA). (D) Bar chart of BSA quantity in (C). (E) SDS-PAGE image of the adsorption from pure BSA and MB solutions, as well as from competitive (BSA/MB) and sequential (BSAMB) adsorption with replicates. (F) Quantity of the adsorbed BSA and MB in (E).
13
Figure 1
14
Figure 2
15