This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.
Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.
Western blotting is a technique that is commonly used to detect and quantify protein expression, including in tissue homogenates or extracts. Over the years, this technique has led to many advances in both basic and clinical research, where it can be used as a diagnostic tool to identify the presence of disease1,2. Western blotting was first described in 1979 as a method to transfer proteins from polyacrylamide gels to nitrocellulose sheets and subsequently visualize proteins using secondary antibodies that were either radioactively labelled or conjugated to fluorescein or peroxidase3. Through the development of commercially available kits and equipment, Western blotting methods have been increasingly standardized and simplified over the years. Indeed, the technique is now readily performed by scientists with varying backgrounds and levels of experience. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. It is important, therefore, that the accessibility of standardized Western blotting methods does not obscure the need for careful experimental planning and design. Experimental considerations include, but are not limited to, sample preparation and handling, selection and validation of antibodies for protein detection, and gel-to-membrane transfer efficiency of particularly small or large (<10 or >140 kDa) proteins4,5,6,7,8,9. Protein quality of the original sample plays a significant role in determining the outcome of the subsequent Western blot analysis. As protein can be extracted from a wide variety of samples and sources, including cell lines, tissues from animal models, and post-mortem human tissues, consistency in handling and processing is required to obtain reproducible results. For example, when long-term storage of samples for protein extraction is required, it is important to realize that, although protein is generally stable at -80 °C, differences in protein stability between extracted proteins and intact tissues at -80 °C have been reported10. Moreover, to obtain reproducible estimates of protein quantities, consistent homogenization of samples is crucial. Optimizing different lysis buffers and homogenization methods (e.g., manual homogenization compared to automated methods) may be required before starting a large-scale quantitative experiment.
Normalization strategies to correct for protein loading and quantification variability are essential to obtain robust, quantitative results of protein expression. Housekeeping proteins such as β-actin, α-tubulin, β-tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have traditionally been used to normalize protein levels for quantification. However, normalization to specific housekeeping proteins for quantification purposes has been increasingly criticized over the past few years11,12. For example, the expression of housekeeping proteins can change across different developmental stages13,14, across tissues from the same animal4, and under various disease conditions4,15,16,17. Therefore, the use of specific housekeeping proteins limits the possibilities of making more complex comparisons between protein expression from different tissues, at different timepoints and under varying experimental conditions. An alternative to housekeeping proteins to control for protein loading variation is the use of a total protein stain (TPS) that labels and visualizes all proteins present in a sample. TPS allows signal normalization based on total protein load rather than levels of one specific protein and therefore quantification of TPS signal should be comparable and reproducible regardless of experimental condition, sample type or developmental timepoint. Examples of total protein stains include Ponceau S, stain-free gels, Coomassie R-350, Sypro-Ruby, Epicocconone, Amydo Black, and Cy5 (reviewed in ref. 18). Each of these methods has specific advantages and limitations and method selection depends on the time and tools available as well as the experimental setup4,18.
In addition to using a TPS to correct for within-membrane loading and quantification variability, it may be necessary to compare samples between different membranes, particularly when performing large-scale protein expression analysis. However, variability in factors such as antibody binding efficiency and total protein stain intensity may introduce further variability between protein samples that are analyzed on separate gels and membranes. For robust quantification in this situation, it is therefore necessary to introduce a further normalization step to account for between-membrane variability. This can be achieved by including an internal loading standard on each of the separately analyzed membranes that is kept constant across experiments. This standard can take the form of any protein lysate that can be obtained in sufficient quantities to be used across all membranes included in the experiment. Here, we use a lysate of mouse brain (obtained from 5 day old control mice), as brain is readily homogenized and the obtained protein lysate contains a significant amount of protein at a high concentration. Loading an internal standard in triplicate allows samples on separate membranes to be normalized and compared directly.
Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints19. By combining a fluorescent TPS with the use of an internal loading standard, we were able to overcome existing limitations in the number of samples and experimental conditions that can be compared within a single experiment. This approach expands the use of traditional Western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.
Tissues for this procedure were obtained from animal studies that were approved by the internal ethics committee at the University of Edinburgh and were performed in concordance with institutional and UK Home Office regulations under the authority of relevant personal and project licenses.
NOTE: This protocol has been optimized using standardized, commercially available kits and reagents in order to increase reproducibility (see Table of Materials).
1. Preparation of samples
2. Gel electrophoresis of protein samples
3. Protein transfer
NOTE: Protein transfer in this protocol is performed using a commercially available semi-dry blotting system (see Table of Materials) for fast and consistent outcomes.
4. Total protein staining
NOTE: Using fluorescent detection provides a substantial benefit over more traditional approaches (e.g., ECL detection), as the linear range and sensitivity can be much better controlled4. Therefore, in steps 4 and 5, a fluorescent TPS and fluorescent secondary antibodies are used (see Table of Materials).
5. Blocking, antibody incubation and detection
6. Western blot analysis and quantification
NOTE: These recommendations are based on the freely available Image Studio software. However, comparable analyses can also be done using other software packages, such as ImageJ.
7. Statistics
We include examples of the use of TPS and an internal standard to facilitate comparisons of protein levels across tissues and time points. Figure 1 shows results from Western blotting on protein extracted from tissues obtained from neonatal (postnatal day 5) in comparison to adult mice (10-week old). TPS and Smn immunoblot are shown in Figure 1A, C. Quantification of fluorescence intensity of the TPS was achieved by measuring the fluorescence intensity inside the rectangle box on each lane and its results are shown in the tables in Figure 1B and 1D. Note that samples from different tissues are characterized by different TPS protein band patterns and therefore it is necessary to use the whole lane for normalization purposes. Indeed, when whole lanes are analyzed, the fluorescence intensity remains relatively similar across samples, indicating TPS for normalization is suitable for this purpose. An internal standard consisting of a P5 brain lysate mixture was also included to illustrate how it can be used for further comparisons between different membranes. Furthermore, in Figure 2, we show how a fluorescent TPS can be used to compare protein levels at different developmental time points. Here, we show Smn levels in brain lysates from neonatal (P5), weaning age (P20) and adult (10W) mice (Figure 2A). Although Smn levels clearly decrease with age, TPS quantification remains constant as illustrated in Figure 2B.
Figure 1. Western blots showing TPS and Smn protein levels in mouse tissues at two different ages. TPS and Smn protein for P5 (A) and 10 week-old (C) mice. (B, D) The fluorescence intensity of whole-lane TPS was calculated and is indicated (in arbitrary units). M: marker/protein standard; kDa: kilodalton; a.u.: arbitrary unit; P5: postnatal day 5; 10W: 10 weeks. Please click here to view a larger version of this figure.
Figure 2. Analysis of Smn expression in mouse tissues at different developmental time points. (A) Brain lysates from tissue obtained from P5, P20 and 10 week-old mice was analyzed using TPS (top panel) and SMN (bottom panel). (B) The fluorescence intensity of the TPS was calculated and is indicated in arbitrary units. M: marker/protein standard; kDa: kilodalton; a.u.: arbitrary unit; P5: postnatal day 5; P20: postnatal day 20; 10W: 10 weeks. Please click here to view a larger version of this figure.
Tissue | Approx. weight* | 1xPBS wash (4 °C) | Repeat wash | Homogenizing buffer ** |
Spinal cord | 40 mg | 150 mL | 3x | 100 mL |
Muscle (GC) | 20 mg | 150 mL | 3x | 100 mL |
Brain | 240 mg | 400 mL | 4x | 400 mL |
Heart | 60 mg | 400 mL | 4x | 200 mL |
Liver | 130 mg | 400 mL | 4x | 400 mL |
Kidney | 45 mg | 400 mL | 4x | 200 mL |
* These weights are indicative values for tissue obtained from P8 mice. | ||||
** These volumes are indications and can be further adjusted according to the weight of the tissue. |
Table 1. Overview of expected tissues weights and corresponding recommendations for PBS washes and lysis buffer volume to be used for homogenization. The weights are indications for tissue obtained from postnatal day 8 (P8) mice. PBS and lysis buffer volumes can be scaled up and down according to experimental needs.
With the appropriate experimental design, control measures and statistical analysis, western blotting can be used to make reliable quantitative estimates of protein expression within and between a varied range of biological samples. The protocol we describe in the current manuscript aims to serve as a guideline for researchers looking to use Western blotting to undertake quantitative analysis across larger and more complex groups of samples, by using a combination of fluorescence-based detection methods, total protein loading normalization and internal standards. Although the focus here is on determining and comparing protein expression from different mouse tissues and at different ages, this approach can also be extended to compare protein expression in other experimental conditions.
A central step in our current protocol is the normalization of proteins of interest to total protein loaded by quantifying a fluorescent total protein stain. TPS normalization corrects for variation in sample loading and error margins in protein quantification methods. However, because the number of protein samples that can be analyzed on a single membrane is often limited, further normalization may be required to compare multiple membranes. Indeed, variability between how proteins are detected on different membranes (due to for example antibody incubation time or temperature variation) may cause variation beyond that introduced through loading and quantification steps of the protocol. Here, we use an internal standard that consists of a single protein lysate mix that is loaded in triplicate on each gel and allows for comparison and normalization between gels/membranes. Theoretically, this could be any protein sample, as long as it can be made in sufficient quantities so that the same sample can be used for all experiments. In our experiments, we use a mixture of brain protein lysates, as brain homogenates contain large quantities of protein and are typically obtained at a high concentration. Averaging quantification of triplicate standard should further increase the accuracy of quantifications across membranes and contribute to the reproducibility of the experiment.
Protein levels can be determined by a number of different techniques and the preferred method depends on the sample type being analyzed and the goal of the experiment. Reproducible quantification of Western blot works best in situations where experimental conditions can be well-controlled, such as when using mouse or other animal models of a defined genetic background. In contrast, in many experiments using human patient samples, this may be less feasible as age, genetic variability and tissue sampling times are much harder to control than in (animal) model systems. Plate-based techniques such as ELISA might be more suitable for these analyses, although the careful validation of antibody specificity is crucial. For example, in fragile X syndrome research, antibodies have been shown to detect different isoforms and when used in ELISA this would lead to overestimation of the total amount of protein as ELISA determines a signal for all isoforms combined24. Optimal choices for methods to determine protein expression are therefore depending on context, sample type and the research question that is being investigated.
Performing adequate statistical analysis is a prerequisite for the reliability of any conclusions drawn from the quantification of biological data. Statistically analyzing complex data as generated by comparing different tissues, time points or other experimental conditions and combinations thereof may require more advanced statistical modelling than ANOVA with post-hoc testing can deliver. For more complex statistical modelling, such as the mixed-effects model approach, we describe in the current manuscript, it may be advisable to seek further advise from biostatisticians. Adequate statistical analysis of large-scale protein expression can greatly improve the robustness of the outcomes and the reliability and reproducibility of results.
In summary, the experimental approach we describe here provides a robust and reproducible method for researchers that want to determine protein expression using western blot in complex samples allowing to answer new and exciting research questions.
The authors have nothing to disclose.
E.J.N.G. is supported by the Wellcome Trust (grant 106098/Z/14/Z). Other funding has been provided by the SMA Trust (SMA UK Consortium; T.H.G. & Y-T.H.), SMA Europe (T.H.G, D.v.D.H. & E.J.N.G.), the University of Edinburgh DTP in Precision Medicine (T.H.G., L.L. & A.M.M.), and the Euan MacDonald Centre for Motor Neurone Disease Research (T.H.G).
Fine Tipped Gel Loading Tips | Alpha Laboratories | GL20057SNTL | |
Halt Protease Inhibitor Cocktail, EDTA-free 100x 5mL | ThermoFisher Scientific | 78437 | |
Handheld homogeniser | VWR Collection | 431-0100 | |
iBlot 2 Gel Transfer Device | ThermoFisher Scientific | IB21001 | |
iBlot Transfer Stack, PVDF, regular size | ThermoFisher Scientific | IB401031 | |
Image Studio Lite | Licor | N/A | Free download from https://www.licor.com/bio/products/software/image_studio_lite/ |
IRDye 800CW secondary antibodies | Licor | — | Select appropriate secondary antibody that is specific against host of primary antibody. |
Micro BCA Protein Assay Kit | ThermoFisher Scientific | 23235 | |
Novex Sharp Pre-stained Protein Standard | ThermoFisher Scientific | LC5800 | |
NuPAGE 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well | ThermoFisher Scientific | NP0323BOX | |
NuPAGE LDS Sample Buffer (4X) | ThermoFisher Scientific | NP0007 | |
NuPAGE MOPS SDS Running Buffer (20X) | ThermoFisher Scientific | NP0001 | |
Odyssey Blocking Buffer | Licor | 927-40000 | |
Purified Mouse anti-SMN (survival motor neuron) monoclonal antibody | BD Transduction Laboratories | 610646 | Is used extensively in the SMN/SMA literature and gives consistent results regardsless of lot number |
REVERT Total Protein Stain, 250 mL | Licor | 926-11021 | |
REVERT Wash Solution | Licor | 926-11012 | |
RIPA Lysis and Extraction Buffer | ThermoFisher Scientific | 89900 | |
XCell SureLock Mini-Cell | ThermoFisher Scientific | EI0001 |