Abstract. Classical Western blotting is considered a qualitative method; however, it can be used for quantitative analysis of proteins pro- vided that specific ...
Methods and considerations for quantitative Western blotting using SuperSignal® Chemiluminescent Substrates Mahesh Mathrubutham and Krishna Vattem
Pierce Protein Research Application Note # 12 AN0012.1
October 2005
Abstract Classical Western blotting is considered a qualitative method; however, it can be used for quantitative analysis of proteins provided that specific controls are included. Using SuperSignal® West Dura Chemiluminescent Substrate we qualitatively and quantitatively detected changes in the regulation of IkBα and p53 in A431 cells treated with EGF (and doxorubicin or cisplatin) by comparing to a standard curve generated using pure protein. ELISAs for both proteins enabled confirmation of the Western data. Additionally, using an imager provided greater linear range than film and, therefore, greater flexibility. SuperSignal West Substrates are therefore useful for qualitative and quantitative Western blotting when the proper controls are used.
Introduction Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE)1 and Western blotting2 are two important and well-known methods for protein characterization. Typically, complex protein mixtures are separated by molecular weight using SDS-PAGE and then electrophoretically transferred to a membrane such as nitrocellulose or polyvinylidene fluoride (PVDF). The proteins on the membrane are probed with an antibody specific for a target protein. If the protein is present then the interaction between the specific antibody and target can be detected using a variety of different methods, such as chemiluminescence,3 fluorescence4 or radioactivity.5 Although classical Western blotting is typically used for qualitative purposes, it also can be used for quantitative analysis of proteins6-8 provided that specific controls are included. In this report we detail the qualitative and quantitative analysis of changes in IkBα and p53 levels in A431 cells treated with epidermal growth factor (EGF) and doxorubicin or cisplatin using SuperSignal West Dura Substrate. We obtained purified target protein for use as an internal control and to create a standard curve. The sample must contain sufficient target protein such that its amount was within the standard curve range. To convert band intensity into a quantitative measurement, the Western blot was analyzed densitometrically using a charge-coupled device (CCD) camera and imaging system. Finally, an ELISA was used to confirm the trends observed by Western blot.
Materials and Methods Cell Culture
For IκBα analysis A431 cells were grown to 90% confluence in DMEM supplemented with 10% fetal calf serum (FCS). The cells were grown using low serum conditions for 24 hours and then treated with epidermal growth factor (EGF; 10 ng/ml) in DMEM with 0.2% FCS. Cells were collected at 5 min, 30 min and 24 hr after adding EGF and lysed using 500 µl of T-PER® Tissue Protein Extraction Reagent (Product No. 78510) with 5 µl of Halt™ Protease Inhibitor Cocktail (Product No. 78410), 1 mM NaF and 1 mM NaVO4. The lysates were stored at -80oC. For P53 analysis A431 cells were grown to 80% confluence in DMEM supplemented with 10% FCS and 1X pen/strep in 10 cm culture dishes at 37oC and 5% CO2 containing humidified incubator. Cells were treated with either 50 µM cisplatin (Cis) or 5 µg/ml of
doxorubicin (Dox) for one hour and subsequently washed one time with phosphate buffered saline (PBS) before supplementing with more DMEM medium. Cells were collected at 4, 8, 20 and 30 hours post treatment and washed two times with cold PBS before lysate preparation. Cells were lysed by incubating the plates for 15 min on ice with 250 µl of M-PER® Mammalian Protein Extraction Reagent (Product No. 78503) containing Halt™ Protease Inhibitor Cocktail (Product No. 78410) and 1 mM EDTA. To obtain maximal yields of cellular proteins the cell lysate was sonicated also. Lysates were clarified by centrifugation and the supernatant stored as aliquots at 70oC. Protein amount was estimated using the Micro BCA™ Protein Assay (Product No. 23235). Quantitative Western Blotting
Pure protein or cell lysates were prepared in Non-reducing Lane Marker Sample Buffer (Product No. 39001), boiled for 5 min and 10 µl per lane were applied to 4-12% polyacrylamide bis-tris gels. A standard curve was included with each Western blot. The linear range for IκBα and p53 was determined using amounts of recombinant IkBα (Upstate Biotechnology, Lake Placid, NY) from 0.0015 to 50 ng and recombinant p53 (Active Motif, Carlsbad, CA) from 1.8750 to 60 ng. The gels were electrophoresed at 120 volts for 60 min and then the proteins were transferred to PVDF membrane (Product No. 8818) by wet electroblotting for 90 min. Blots were blocked with StartingBlock™ Blocking Buffer (Product No. 37542) for 60 min at room temperature and then incubated overnight at 4oC with antiIκBα (Upstate Biotechnology, Lake Placid, NY) or anti-P53 (Active Motif, Carlsbad, CA) antibodies at 1 µg/ml prepared in StartingBlock™ Blocking Buffer. Blots were washed three times for 10 minutes each with PBS containing 0.05% Tween-20 (PBS-T). Blots were incubated with goat anti-rabbit antibody (1:1,000 dilution from 10 µg/ml stock; Product No. 1858415) for 60 min at room temperature and then washed four times in PBS-T for 15 min each. Signal detection was achieved using SuperSignal West Dura Substrate (Product No. 34075). Blots were imaged using the Kodak 20000MM Image Station, and protein band densities were analyzed using the spot density analysis software provided with the image station. The quantitative Western blot data were confirmed using p53 and IκBα ELISA assays (Assay Designs, Ann Arbor, MI). The ELISA assays were performed according to the manufacturer’s instructions.
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Application Note # 12
Results and Discussion For quantitative Western blot methods, a pure form of the target protein must be available to generate a standard curve in each blot to quantify unknown samples. Densitometric analysis of the Western blots indicated that the linear range for IκBα was 0.097-3.1250 ng (Fig. 1) and for P53 was 1.8750-60 ng (Fig 2). This range of pure protein samples also served as an excellent internal positive control and counteracted variations in Western blotting efficiency.
In the Western blot analysis, levels of IκBα remained constant for the first 5 min after EGF treatment, rapidly dropped by 30% after 30 min and finally gradually increase during the next 24 hr (Fig. 3). Densitometric analysis of the p53 Western blot (Fig. 4) revealed that the levels of p53 changed slightly over the first 8 hours after treatment with Dox and then rapidly increased during the next 20-30 hours. Treatment with Cis resulted in a gradual increase in p53 for 30 hours. 1
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Figure 1. Linear range determination for recombinant IκBα by Western blot analysis. Panel A: Serial dilutions of recombinant IκBα (50-0.1 ng) were separated by electrophoresis, transferred to PVDF membrane and Western blotted. Recombinant IκBα was detected using SuperSignal West Dura Substrate. Panel B: Band intensities on the blot were analyzed by spot densitometry and then plotted against IκBα amount to generate a standard curve within the linear range of IkBα.
Figure 3. Quantitation of IkBα in EGF treated A431 cell lysates by quantitative Western blotting. Panel A: Known amounts of recombinant IkBα (rIkBα) were Western blotted to generate a standard curve. Lanes 1-6 contain 0.15-0.012 ng rIkBα. Panel B: Lysates of EGF treated A431 cells were Western blotted and probed for IkBα. Cells were treated with EGF as follows: non-treated (lanes 1,2), 50 ng for 5 minutes (lanes 3, 4); 50 ng for 30 minutes (lanes5, 6); and 50 ng for 24 hours (lanes 7, 8). Panel C: 2 Recombinant IkBα Standard curve (r = 0.99). Panel D: Variation in the amount if IkBα with EGF exposure time as determined by quantitative Western blotting.
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Figure 2. Linear range determination for recombinant p53 by Western blot analysis. Panel A: Serial dilutions of recombinant p53 (60-1.875 ng) were separated by electrophoresis, transferred to PVDF membrane and Western blotted. Recombinant p53 was detected using SuperSignal West Dura Substrate. Panel B: Band intensities on the blot were analyzed by spot densitometry and then plotted against p53 amount to generate a standard curve within the linear range for p53.
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Figure 4. Quantitation of p53 in Dox and Cis treated A431 cell lysates by quantitative Western blotting. Panel A: Known amounts of recombinant p53 (rp53) were Western blotted to generate a standard curve. Lanes 1-6 contain 60-1.875 ng rp53. Panel B: Lysates of Dox and Cis treated A431 cells were Western blotted and probed for p53. Cells were treated as follows: non-treated (lane 1); Dox for 4 hours (lane 2); Dox for 8 hours (lane 3); Dox for 20 hours (lane 4); Dox for 30 hours (lane 5); Cis for 4 hours (lane 6); Cis for 8 hours (lane 7); Cis for 20 hours (lane 8); Cis for 30 hours (lane 9) and 5.6 ng p53 (lane 10). Panel C: Recombinant p53 2 Standard curve (r = 0.99). Panel D: Variation in amount of p53 with Dox and Cis exposure time as determined by quantitative Western blotting.
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Application Note # 12 The trend in IkBα and p53 levels was confirmed by ELISA (Fig. 5) and also correlated well with published data.9,10 With respect to p53, even though the ELISA confirms the Western blot data, closer comparison of the Western blot and ELISA data revealed that the Western blot was more sensitive to changes in the quantity of p53 between 20 and 30 hr (Fig. 5). At the 20-30 hr time point there was a large amount of p53 present, but the ELISA did not detect the gradual increase of p53 between these time points, whereas the Western blot did detect a gradual increase. In contrast, changes in p53 during the first 8 hours when levels of p53 are relatively low were detected better by ELISA than by quantitative Western blot. Therefore, the ELISA is more sensitive to changes in p53 at low levels while the Western is more sensitive to changes in p53 at high levels.
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References 1. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685. 2. Towbin, H., et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350-4354. 3. Bronstein, I., et al. (1992). Improved chemiluminescent western blotting procedure. Biotechniques 12: 748-753. 4. Diamandis, E.P., et al. (1992). Quantitative western blot analysis and spot immunodetection using time-resolved fluorometry. J. Immunol. Methods. 147: 251-259. 5. Hunger, H-D., et al. (1994). Quantitative Western blotting using [γ33 P]ATP and the ultrasensitive bio-imaging analyzer. Anal. Biochem. 217: 98-102. 6. Schiavini, D.G., et al. (1989). Quantitative Western immunoblotting analysis in survey of human immunodeficiency virus-seropositive patients. J. Clin. Microbiol. 27: 2062-2066. 7. Martin, P-Y., et al. (1999). Seelective V2-receptor vasopressin antagonism decreases urinary Aquaporin-2 excretion in patients with chronic heart failure. J. Am. Soc. Nephrol. 10: 2165-2170. 8. Feissner, R., et al. (2003). Chemiluminescent-based methods to detect subpicomole levels of c-type cytochromes. Anal. Biochem. 315: 90-94. 9. Sun, L., and Carpenter, G. (1998). Epidermal growth factor activation of NF-kappaB is mediated through IkappaBalpha degradation and intracellular free calcium. Oncogene. 23: 2095-2102. 10.Kwok TT, et al. (1994). Up-regulation of a mutant form of p53 by doxorubicin in human squamous carcinoma cells. Cancer. Res. 54: 2834-2836. 11.Sato, M., et al. (2002). Quantification of Galectin-7 and its localization in adult mouse tissue. J. Biochem. 131: 255-260. 12.Xing, C., and Imagawa, W. (1999). Altered MAP kinase (ERK1,2) regulation in primary cultures of mammary tumor cells: elevated basal activity and sustained response to EGF. Carcinogenesis. 20: 12011208.
Figure 5. Quantitation of IkBα and p53 in EGF, Dox and Cis treated A431 cells by ELISA.
This report demonstrates that quantitative Western blotting can be performed using SuperSignal Chemiluminescent Substrates; however, the quantity of IkBα and P53 determined by Western blotting and ELISA differs. Even though both Western blotting and ELISA rely upon immunodetection, they are different methods and as such are developed using specific antibodies and recombinant controls. Therefore, variation in absolute protein amounts may be caused by differences in the recombinant protein used for controls, primary antibody specificity, and variations in protein interactions within each technique.
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Although Western blotting is often considered qualitative, it also can be advantageous as a quantitative method. Quantitative Western blotting can be used for biological samples where no ELISA is available. Quantitative Western blotting could also be applied in situations where certain components in a biological sample may interfere with an ELISA. Often antibodies directed against one protein may interact with equal specificity with another closely related protein. In such situations an ELISA may generate false positives or overestimation of the target protein abundance. Because Western blotting involves gel electrophoresis, variations in molecular weight can be exploited to distinguish and quantify the target protein alone.11, 12
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