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  • 1. Journal of Immunological Methods 345 (2009) 40–48 Contents lists available at ScienceDirect Journal of Immunological Methods j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j i m Research report Improved semiquantitative Western blot technique with increased quantification range F. Heidebrecht a,⁎, A. Heidebrecht b, I. Schulz a, S.-E. Behrens c, A. Bader a a Cell Techniques and Applied Stem Cell Biology, Biocity, University of Leipzig; Deutscher Platz 5, 04103 Leipzig, Germany b DLR, Braunschweig, Germany c Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Germany a r t i c l e i n f o a b s t r a c t Article history: With the development of new interdisciplinary fields such as systems biology, the quantitative Received 11 February 2009 analysis of protein expression in biological samples gains more and more importance. Although Accepted 31 March 2009 the most common method for this is ELISA, Western blot also has advantages: The separation of Available online 5 April 2009 proteins by size allows the evaluation of only specifically bound protein. This work examines the Western blot signal chain, determines some of the parameters relevant for quantitative Keywords: analysis and proposes a mathematical model of the reaction kinetics. Using this model, a Semiquantitative Western blot Calibration curve semiquantitative Western blot method for simultaneous quantification of different proteins Hyperbolic regression using a hyperbolic calibration curve was developed. A program was written for the purpose of hyperbolic regression that allows quick determination of the calibration curve coefficients. This program can be used also for approximation of calibration curves in other applications such as ELISA, BCA or Bradford assays. © 2009 Elsevier B.V. All rights reserved. 1. Introduction chemiluminescence images has become higher than that of X- ray film (Feather-Henigan et al., 1999). The Western blot or immunoblot is a detection method of An even higher sensitivity can be reached using the specific proteins in biological samples. The first attempt to quantum dot fluorescence technology, and also a multiplex immobilize the proteins on a membrane has been described Western blot with two colors of quantum dots is possible by Renart et al. (1979). The use of nitrocellulose membranes (Ornberg et al., 2005). was first described 1979 by Towbin et al. (1979). The name of Nowadays Western blots are used either for absolute the technique was introduced by Burnette (1981). quantification in combination with a calibration curve of the Although Western blot was considered rather a qualitative recombinant protein of known concentration (see Mathrubu- method, first attempts to quantify the results were reported tham M. and K. Vattem: http://www.piercenet.com/files/ already in 1985 (Lin et al., 1985). AN0012ln5.0.pdf), or for quantification of samples relative to The efficiency of the Western blot was improved with the a control sample. For a relative quantification a house- discovery of the chemiluminescence technique, that allows an keeping gene (Chen et al., 2006; Roman-Blas et al., 2007; increased sensitivity and higher stability of the detection Kim et al., 2008), or a staining of the transferred proteins on signal. Another advantage of this method is the use of non- the membrane (Chen et al., 2008) is used to normalize the radioactive reagents (Kricka, 1991). Through the development data. of new technologies for data acquirement such as chemilu- For a quantitative analysis of Western blots, a correlation minescence systems with a cooled CCD camera, the quality of between the measurable results and the amount of protein in the examined samples must be established. This correlation is determined by the different processing steps: Western blot, ⁎ Corresponding author. Tel.: +49 341 9731362; fax: +49 341 9731359. primary and secondary antibody binding kinetics, chemilu- E-mail address: felicia.heidebrecht@yahoo.de (F. Heidebrecht). minescence reaction, image acquirement and processing, 0022-1759/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2009.03.018
  • 2. F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 41 quantification of the image (densitometry). Parameters that in 5% acetic acid, or with AmphiGreen Fluo (Biostep, Jahnsdorf influence the correlation are: blot transfer conditions, blot Germany) according to manufacturer's protocol. irregularities (Schilling et al., 2005), concentration of anti- bodies and ECL substrate, exposure time, camera sensitivity, 2.3.3. Antibodies color format and adjustments, band definition and back- In order to be able to investigate proteins of different sizes ground correction. on the same membrane, the membrane was cut into This paper analyses some critical aspects of the Western horizontal stripes, see Fig. 2. The stripes were probed using blot procedure with focus on the kinetics of the antibodies the Western blot protocol from Cell Signaling Technology and ECL reaction. The systematic analysis of the different (http://www.cellsignal.com/support/protocols). parameters allows the derivation of a mathematical model of The antibodies against phosphorylated STAT3 (Tyr 705), the kinetics. This is then used to determine the calibration phosphorylated Akt (Tyr 308) and phosphorylated ERK1/2 curve type and to address some sources of inaccuracies in the (Thr 202/Tyr 204) and secondary horseradish peroxidase- protocol. A program was written that determines the coupled antibodies were from Cell Signaling Technology. coefficients of the calibration curve. 2.3.4. Chemiluminescence 2. Materials and methods The immunoblots were incubated with enhanced chemi- luminescence solution from Cell Signaling Technology for 2.1. Materials 1 min and exposed between 30 and 120 s on a Molecular Imager ChemiDoc XRS System from Bio-Rad which has a William's medium E (WME), L-glutamine and dexametha- cooled and calibrated CCD sensor with a linear response sone were obtained from Sigma-Aldrich (Deisenhofen, Ger- curve. Constant substrate concentration was achieved by many), fetal calf serum (FCS) from Biochrom (Berlin, taking the pictures while the membrane is completely Germany), penicillin/streptomycin (10,000 U/ml) from submerged in ECL solution. Gibco (Paisley, Scotland) and Interleukin-6 from Immuno- ECL images were taken in the native format using the tools GmbH (Friesoythe Germany). system's standard software Quantity One and then exported to 16-bit TIFF format. 2.2. Sample preparation 2.3.5. ECL image analysis For the Western blots described in this paper, protein All images were treated in a way that avoids loss of extracts from mouse hepatocytes were used. The isolation information. This means: No use of lossy image formats such and culture of the cells is described in the Supplementary as JPEG; no change of resolution; always use the highest information. The protein extracts were prepared as follows: possible color depth (32 or 16 bit) to preserve precision; no The medium was completely removed, the cells were washed overexposure when taking pictures to avoid highlight clip- twice in ice-cold PBS and lysed with 500 μl lysis buffer per ping and no change to contrast and/or brightness that would well (25 mM Tris, 150 mM NaCl; pH 7.2) supplemented with result in clipped highlights or shadows; also, no “Gamma” protease and phosphatase inhibitor cocktails (Sigma- correction, as this would destroy the linearity of the images. Aldrich). Cells were scraped off on the well and lysates were Exceptions were only made for illustration purposes or in the transferred to microcentrifuge tubes and kept on ice. During form of non-destructive layers within Photoshop®, in order to incubation (30 min), the samples were sonicated for 10–15 s. increase visibility of some image features. Lysates were centrifuged at 13,000 rpm at 4 °C for 20 min in a Analysis of ECL images was performed using the public table centrifuge to remove cell debris. The cleared super- domain ImageJ program (developed at the National Institutes natants were transferred to fresh tubes and protein concen- of Health and available at http://rsb.info.nih.gov/ij/), using trations were determined by BCA assay (Pierce, Rockford, the “Gel Analysis” functions. Background correction was done USA). using a “rolling ball” method with a radius of 4 times the The reference sample for the calibration curve was width of a band. Result of the analysis is a value for each band obtained from 25 ⁎ 106 freshly isolated hepatocytes stimu- which is proportional to the Integrated Density Value (IDV) of lated in suspension with 20 ng/ml IL-6 for 30 min. that band. 2.3. Western blot 2.3.6. Membrane quantification If a quantitative analysis of membrane staining was done, 2.3.1. SDS-PAGE the image of the stained membrane, as well as a reference Total cellular lysates (20 μg) and a calibration curve of picture of a white sheet of paper (both with the same lighting different protein amounts of the reference sample were and aperture setting), were linearised, using the DRI loaded on a 10% SDS-polyacrylamide gel. (Dynamic range increase) technique: Exposure series were taken instead of single images and converted to a linear HDR 2.3.2. Blot transfer (High Dynamic Range) picture (Debeverec and Malik, 1997). The separated proteins were transferred to 0.45 μm HDR conversion was done using the free software Picturenaut nitrocellulose membrane in cold transfer buffer (48 mM (available from http://www.picturenaut.de). Other software, Tris, 39 mM glycine, 1.39 mM SDS, 20% methanol) at a such as HDRShop and Photomatix, is also freely available and constant 45 mW/cm2 for 15 min using a semi dry blot provides equivalent functionality. After linearisation, vignet- chamber. The transfer was assayed either with 0.1% Ponceau S ting and light distribution were corrected by dividing the
  • 3. 42 F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 pixel values of the membrane image by those of the reference The membrane is then incubated with the primary image. This was done directly on the HDR images using the antibody. Some authors suggest that the kinetics of immu- free, open source software Blender (http://www.blender. noassays is hyperbolic (Studnika, 1987; Braitbard et al., 2006). org). ImageJ provides the same functionality. The resulting Indeed the reaction between antibody and antigen can be image was then converted to 16 bit grayscale and quantified described by the law of mass-action (Ekins and Gosling, 1970; using Adobe® Photoshop®, using a constant background O'Connor and Gosling, 1997; Holland and Holland, 2003) that value. The IDV was determined using the “average” filter on shows similarities to the hyperbolic enzyme kinetics pro- each area of interest, subtracting the background value from posed by Michaelis and Menten (1913). Thus, antibodies can the result and multiplying the remainder by the size of the be assumed to have a hyperbolic binding behavior. Eq. (1) area of interest. This is mathematically equivalent to adding shows a general hyperbolic equation: the single pixel values in the area.   a CAB1 = f Cprot = CAB1;max − ; ð1Þ Cprot − Cprot;0 2.3.7. Calibration curve After quantification of the ECL bands, a hyperbolic where CAB1 is the local concentration of bound primary regression was done on the results from the dilution series, antibody and Cprot the concentration of the protein. CAB1,max is linking the IDVs to the amounts of protein in the dilution the theoretical maximum saturated concentration of bound series samples. This was done using the Python script that is antibody. a and Cprot,0 have no direct physical significance but part of the Supplementary material to this paper. The are coefficients that describe the curvature of the hyperbola. resulting curve was inverted and used to compute an The secondary antibody binds to the primary antibody in equivalent protein amount for each IDV value of the sample the same fashion, i.e. with hyperbolic saturation. It can be bands. These values are then proportional to the amount of shown (see Supplementary information) that a hyperbolic specific protein in the band. A graphical representation of the function of another hyperbolic function is again a hyperbolic complete data processing is in Fig. 5. curve, with different coefficients, thus the combined transfer function of two chained antibodies is still hyperbolic. Fig. 1 shows the influence of a variation in antibody concentrations 3. Results and confirms that in case of visible saturation the behavior can indeed be represented by a hyperbola. 3.1. Western blot signal chain The ECL reaction is an enzyme–substrate reaction, where the substrate is available in abundance and the enzyme is As in any quantitative assay, a standard curve is necessary bound to the secondary antibody. The local light emission rate also in Western blot. From a certain protein amount the curve (or intensity, I) is therefore dependent on the local concen- becomes non-linear and saturation occurs. A quantitative tration of enzyme Cenz (which is proportional to CAB2) and the determination of proteins in samples is usually done only in enzyme's reaction rate: the linear range of the standard curve (Blomberg and Klasse, 1988). I = Cenz · VECL : ð2Þ In order to extend the useful range of the standard curve, The reaction rate VECL is determined by the Michaelis– the type of the standard curve regression must be deter- Menten equation, given that substrate concentration is much mined. For this, it is necessary to regard the complete process higher than enzyme concentration: chain from initial protein concentration in a sample to the measured band intensity in the ECL image. Every step with Vmax · Csubst linear behavior preserves the information on relative protein VECL = ; ð3Þ Km + Csubst amounts, while non-linear effects such as saturation can distort these relations. where Vmax and Km are constants and Csubst is the substrate For each sample, a known quantity of reference protein concentration. It is noteworthy that the reaction velocity is solution is pipetted into the gel slots. A certain fraction of this not dependent on the enzyme concentration but only on the is then transferred to the membrane during the blotting substrate in the ECL solution. If Csubst is constant, the reaction process. Ideally, the amount of protein in the membrane is velocity is also constant over the complete membrane. If the proportional to the amount in the gel. There are however non- membrane is only netted with ECL solution, the substrate linear effects in the blot, caused by non-constant distribution concentration can become inhomogeneous because over of electric resistance depending on local protein concentra- some parts of the membrane the reagent is consumed faster tion, size, temperature and possibly other factors (Schilling than elsewhere. Therefore, the images are taken with the et al., 2005). Since these errors are not systematic, the transfer membrane submerged in ECL solution in order to achieve function is still assumed to be linear in this paper, although constant substrate concentration during the whole image some of the errors can be corrected later in the process (see acquisition process. This way, there is an exchange between Section 3.3). spots with high and low reagent consumption. Fig. 1. Example blot of three times the same dilution series, incubated with different concentrations of antibody. A: Image of membrane stained with Ponceau S. The staining was quantified at the vertical coordinate of P-Stat3 ";. B: Quantification results show a linear function, thus the amount of protein at the vertical coordinate is in fact proportional to the amount of whole protein ";. C: ECL image shows P-Stat3 (Tyr 705) bands ";. D: Quantification result of ECL image. Depending on the combination of antibody concentrations, saturation is visible or not. While hyperbolic regression has the same coefficient of determination in all cases, the linear curve becomes inaccurate if saturation occurs.
  • 4. F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 43
  • 5. 44 F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 Fig. 2. Western blot analysis of several proteins using one calibration curve. A: Membrane staining with Ponceau S. Twelve samples on the left side and 6 samples for the calibration curve on the right side. Horizontal lines mark the places where the membrane was cut. B: ECL image of the different proteins. Like most chemiluminescence systems, the one used for this For the hyperbolic regression, a script was written using the paper has a calibrated sensor and delivers linear output, so scripting language Python (freely available from http://www. the pixel values in the image are proportional to the local light python.org) and the add-on module Scipy (also freely available emission rate I as long as they are not overexposed. The image from http://www.scipy.org). The script takes the protein quantification process then integrates the pixel values over amounts and quantification results for the dilution series sam- each band to give an integral value for the brightness of that ples and constructs a correlation using least-squares regression band which is proportional to the amount of light emitted by with a hyperbolic function, taking an analytical approximation the whole band. (Studnika, 1987) as a starting point and refining it by using the Summing up the complete process chain, the result of ECL Scipy routine leastsq to minimize the sum of error squares. The band quantification is proportional to a hyperbolic function of coefficients for the resulting hyperbola are returned and can the original amount of protein in the samples. be used in any spreadsheet application to linearise the results This hyperbolic function can be linear over the relevant from the other samples. The script itself, hard- and software interval, depending on many factors such as protein amount, requirements and a more detailed description of it are blotting parameters and antibody concentrations, but if a available as part of the Supplementary information. dilution series is used to derive the parameters of the The number of samples in the dilution series must be at standard curve, relative quantification can be done also in least four because the hyperbolic function used has three the non-linear range, up to protein concentrations where degrees of freedom, thus will always match three points with almost full saturation is reached and the calibration curve perfect fit. This is not desirable because in this case, every becomes horizontal. error in each of the samples is directly transferred to the regression curve. The more samples are used the larger the 3.2. Calibration curve statistical significance of the regression. This is comparable to using the mean value of several single values. In this paper, 6 Regarding the composition of the calibration curve, samples were used to derive the standard curve. several aspects are important. While any protein solution can be used, it is helpful to use 3.3. Membrane staining one that contains each of the proteins of interest in sufficient amount. In this study, a protein sample from the same cell type Quantification quality of the membrane staining can be as the one being analyzed was used. As can be seen in Fig. 2, a greatly improved by correcting the membrane images prior to single dilution series can also be used to quantify several the actual quantifications. The procedure is based on the different proteins from one Western blot, if the protein optical and physical properties of the system. composition is similar to that of the samples. The range of the The first step towards a linear staining image is the dilution series should be large enough to include all of the linearisation of the sensor response. This is done by taking an protein concentrations that are expected in the other samples. exposure series (multiple images with different exposure Slight extrapolation outside of the range of the dilution series is lengths) and combining the images into an HDR image possible, but accuracy is reduced with increasing extrapolation (Debeverec and Malik, 1997) which is by definition linear. distance. In order to make sure that the range is sufficient, an When using a light source, illumination is usually not advance test blot can be made with a very large dilution series constant over the complete membrane area. Also, most and only few test samples to determine the lowest and highest camera lenses produce vignetting (i.e. the corners of an dilution that will be needed. image appear darker). Both effects can be corrected by a so-
  • 6. F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 45 Fig. 3. Correction of light distribution and lens vignetting (“flat-fielding”). A: Uncorrected image of a Western blot membrane stained with Ponceau S. B: Reference (“flat-field”) image of a white sheet of paper. C: Corrected image. Brightness values have been adjusted to improve visibility of the brightness gradients in the original and the reference picture. called flat-field correction (Englert and Harlander, 2006). While quantifying the membrane staining is optional, it Fig. 3 illustrates the procedure, and detailed information can can be used for several purposes: be found in the Supplementary information. The flat-field correction used in this paper consists of taking a reference • Controlling the quality of the blot transfer. When using image of a white sheet of paper and dividing the membrane quantified results, it is easier to spot some systematic or image by this reference image. Although for the results in this random errors. paper the software Blender was used, it was found that ImageJ • To some extent: If blotting and/or pipetting errors are is capable of performing the division (via “Process → Math → detected, the results of the ECL quantification may be Divide”) also for HDR images (provided they are in gray scale, corrected by using the numbers from the membrane not RGB mode). Because of the simpler interface, the use of staining as the relative amounts of total protein instead of ImageJ is recommended. a constant value. However, quantification errors for the It has been found that images acquired with fluorescent membrane image appear to be as high as or slightly higher staining are better suited for quantification compared to than those for the ECL image. One reason for this is that the Ponceau S. This is connected to the fact that a color staining background is usually less regular than in ECL images, and such as Ponceau S darkens the membrane locally. Since the background correction using available methods becomes color of the membrane cannot become darker than that of the less accurate. Therefore, staining quantification should only pure staining color, there must be saturation before that color be used to correct ECL results if it is reasonably certain that value is reached. This leaves only a certain useful range of the error being corrected is larger than the quantification staining concentrations, independent of whether the actual error for the membrane image. binding of Ponceau S to the protein is saturated. With • In this paper, the quantitative membrane staining analysis fluorescent staining, the membrane appears brighter with was also used to produce the results shown in Fig. 1, section B. increasing protein concentration, and there is no theoretical upper bound for brightness. Thus, saturation with Amphi- 3.4. Data processing Green Fluo will only occur if the binding itself is saturated. AmphiGreen Fluo has been found to have a larger dynamic Fig. 5 illustrates the data evaluation. All bands in the ECL range (relative intensity quotient between weakest and image (Fig. 5.A) are quantified. This results in a set of numbers strongest bands in the linear range) in all cases regarded. representing the integrated density values from the ECL Fig. 4 shows an example comparison of Ponceau S vs. image for each band. Since for the calibration curve the AmphiGreen Fluo staining. relative amounts of protein are known, the points can be
  • 7. 46 F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 Fig. 4. Comparison between Ponceau S (left) and AmphiGreen Fluo (right) membrane staining on the same membrane. A: Membrane pictures, contrast enhanced to improve visibility. The uniform rectangles mark the areas that were used for quantification. They are equal for both images. B: Quantification results for both stainings. Ponceau S shows saturation while AmphiGreen produces a straight line for the same membrane. drawn into a graph that connects the IDV for each band to the proteins and allows simultaneous analysis of different amount of whole protein that was used for the dilution series. proteins on the same membrane. For these pairs of values, a hyperbolic regression is done A computer program was written for determination of the (Fig. 5.B). The resulting formula then can be used to connect parameters of the hyperbolic regression curve and is available the IDV for each sample to a protein amount (Fig. 5.C). This as part of the Supplementary information. protein amount is the amount of total protein from the Summarizing the steps according to our protocol: dilution series sample that contains the same amount of the • Loading and blotting of samples and calibration curve specific protein. In the case presented here, this value has no • Membrane staining direct meaning but is proportional to the amount of specific • Optionally: staining quantification protein in the sample. It can be normalized by another value, • Cutting the membrane into stripes for different proteins for example from a control sample. If the amount of specific • Blocking and incubation with antibodies, ECL protein in the dilution series was known, the method could • Densitometry of the ECL image with ImageJ or other also be used to derive absolute protein amounts. software If visible irregularities have been found in the membrane • Determination of the hyperbolic regression coefficients staining, it is possible to reduce them by normalizing each using the regression script value by the IDV determined during membrane quantification • Computation of the samples' relative protein content at the respective sample location. This is equivalent to relating according to the calibration curve the amount of specific protein to the amount of whole protein • Normalization of the calculated amounts to a house- on the membrane, thus giving a concentration value that is keeping protein in the same sample. independent of the quality of the blot transfer. As pointed out before, this introduces errors from staining quantification into Our results show that depending on the relative concen- the final result, thus should only be used if blotting errors are tration of the antibody and antigen, saturation is present in large and a repetition of the blot is not practical, and was not the ECL image or not. Therefore, use of a dilution series is used in the Western blots used for this paper. always recommended in order to determine whether the analyzed samples are in the linear range. Performing a 4. Discussion hyperbolic standard curve regression on the dilution series requires no big additional effort and provides valid results We describe a semiquantitative Western blot method with both in the linear and in the non-linear range of the kinetics, a hyperbolic fitting of the standard curve which includes the while being both more accurate and reliable. Since the use of a calibration curve also for relative quantification of Western blot procedure itself is unchanged, the accuracy is
  • 8. F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 Fig. 5. Overview of ECL image quantification. A: A stripe of the blot membrane, containing P-Erk bands from samples as well as that contained in the dilution series. Total protein amounts for the dilution series are given. B: Using the dilution series, a standard curve is derived by hyperbolic regression. The general formula, the regression results, and the resulting function are shown that link the protein amount P to the measured IDV. C: The inverted standard curve, links IDV to the equivalent total protein P′. P′ is proportional to the amount of protein in each of the samples, also if the measured IDVs are not. 47
  • 9. 48 F. Heidebrecht et al. / Journal of Immunological Methods 345 (2009) 40–48 on par to that of other quantitative Western blot methods. In Appendix A. Supplementary data the blots performed for this study, the mean relative error was generally around 10%, and standard deviation was between 10 Supplementary data associated with this article can be and 20%. found, in the online version, at doi:10.1016/j.jim.2009.03.018. The procedure does not compensate for non-systematic (or partially systematic) errors due for example to inhomo- geneities of the blot transfer. A method for this has been References proposed (Schilling et al., 2005). Using the membrane staining to correct such blot irregularities and also pipetting Blomberg, J., Klasse, P.J., 1988. Quantification of immunoglobulin on electrophoretic immunoblot strips as a tool for human immunodefi- errors as described in Section 3.3 could potentially serve a ciency virus serodiagnosis. Journal of Clinical Microbiology 26 (1), 111. similar purpose, but the inaccuracies of the staining quanti- Braitbard, O., Glickstein, H., Bishara-Shieban, J., Pace, U., Stein, W.D., 2006. fication were found to be not much smaller than the errors Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests. Proteome being corrected, so it is at the moment only recommended as Science 4 (12). an emergency measure for large and obvious blotting errors. Burnette, W.N., 1981. “Western blotting”: electrophoretic transfer of proteins The situation could be improved by more reliable quantifica- from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitro- cellulose and radiographic detection with antibody and radioiodinated tion methods for membrane staining, including more accu- protein A. Analytical Biochemistry 112, 195. rate background correction, or by better staining procedures. Chen, E., Kwon, Y.T., Lim, M.S., Dube, I.D., Hough, M.R., 2006. Loss of Ubr1 Using luminescent staining instead of reflective does help, but promotes aneuploidy and accelerates B-cell lymphomagenesis in TLX1/ HOX11-transgenic mice. Oncogene 25, 5725. by itself is not sufficient to make membrane staining a Chen, J., Guerriero, E., Lathrop, K., SundarRaj, N., 2008. Rho/ROCK signaling in universal instrument to reliably increase the overall accuracy regulation of corneal epithelial cell cycle progression. IOVS 49 (1), 175. of the semiquantitative Western blot. Debeverec, P.E., Malik, J., 1997. Recovering high dynamic range radiance maps The quantification method proposed in this paper is based from photographs, SIGGRAPH '97. Proceedings of the 24th Annual Conference on Computer Graphics and Interactive Techniques, p. 369. on the physical properties of the Western blot and therefore is Ekins, R., Gosling, J.P., 1970. Theoretical aspects of saturation analysis. In: able to deal with some of the systematic errors that appear Diczfalusy, E. (Ed.), Karolinska Symposia on Research Methods in when using a simpler approach, most notably antibody Reproductive Endocrinology: Steroid Assay by Protein Binding, p. 11. Englert, C.R., Harlander, J.M., 2006. Flatfielding in spatial heterodyne saturation and errors incurred during image acquisition. spectroscopy. Applied Optics 45 (19), 4583. Being able to include partly saturated bands greatly increases Feather-Henigan, K., 1999. Immunoblot imaging with a cooled CCD camera the range of protein concentrations which can be determined. and chemiluminescent substrates. American Biotechnology Laboratory 17, 44. Taking the optical properties of the camera and lighting Holland, T., Holland, H., 2003. A mathematical model of immunohistochem- system into account, the requirements to the image acquisi- ical preparations, which provides quantitative predictions. Journal of tion system can be reduced. Still, of course, the error Microscopy 214, 1. Kim, T.H., Huh, J.H., Lee, S., Kang, H., Kim, G.I., An, H.J., 2008. Down- correction introduced by using a calibration curve will not regulation of claudin-2 in breast is associated with advanced disease. yield useful results for fully saturated bands, and flat-fielding Histopathology 53 (1), 48. images and using DRI techniques will not neutralize noise, nor Kricka, L.J., 1991. Chemiluminescent and bioluminestcent techniques. Clinical Chemistry 37/9, 1472. make weak bands easier detectable if the camera system is Lin, J.C., Choi, E., Pagano, J.S., 1985. Qualitative and quantitative analyses of not sensitive enough. Epstein–Barr virus early antigen diffuse component by western blotting Hyperbolic saturation behavior is often found in biological enzyme-linked immunosorbent assay with a monoclonal antibody. analytical methods, including antibody and enzyme reactions, Journal of Virology 53, 793. Michaelis, L., Menten, M.L., 1913. Die Kinetik der Invertinwirkung. Biochem- thus the use of a hyperbolic curve for standards curve ische Zeitschrift 49, 339. regression might also benefit the quantitative analysis of O'Connor, T., Gosling, J.P., 1997. The dependence of radioimmunoassay other methods, such as ELISA, Bradford and BCA (see also detection limits on antibody affinity. Journal of Immunological Methods 208, 181. Studnika, 1987). The script provided in the Supplementary Ornberg, R.L., Harper, T.F., Liu, H., 2005. Western blot analysis with quantum information executes a general hyperbolic regression and is dot fluorescence technology: a sensitive and quantitative method for therefore suited for all of these applications. multiplexed proteomics. Nature Methods 2 (1), 79. Renart, J., Reiser, J., Stark, G.R., 1979. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for Acknowledgements studying antibody specificity and antigen structure. Proceedings of the National Academy of Sciences 76 (7), 3116. Roman-Blas, J.A., Stokes, D.G., Jimenez, S.A., 2007. Modulation of TGF-b We thank Dr. Juan Cabello Pardos for reviewing the signaling by proinflammatory cytokinesin articular chondrocytes. manuscript, Mario Keller for help with laboratory work, Osteoarthritis and Cartilage 15, 1367. Andrea Robitzki's lab for sharing their Chemiluminescence Schilling, M., et al., 2005. Computational processing and error reduction strategies for standardized quantitative data in biological networks. FEBS System with us and Michael Sonntag from Biostep for lending Journal 272, 6400. out UV-lights and green filter for testing the AmphiGreen Fluo Studnika, G.M., 1987. Hyperbolic regression analysis for kinetics, electrophor- staining solution. esis, ELISA, RIA, Bradford, Lowry, and other applications. CABIOS 3 (1), 9. This work was partially supported by the German Federal Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some Ministry of Education and Research (BMBF), Projekt System- applications. PNAS 76 (9), 4350. biologie; (grant number 0313081).