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* These authors contributed equally
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. ....
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.......
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.......
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 lo.......
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).
....Name | Company | Catalog Number | Comments |
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 mLÂ | Licor | 926-11021Â | |
REVERT Wash Solution | Licor | 926-11012 | |
RIPA Lysis and Extraction Buffer | ThermoFisher Scientific | 89900 | |
XCell SureLock Mini-Cell | ThermoFisher Scientific | EI0001 |
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