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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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. ....

Protocol

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).

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Representative Results

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.......

Discussion

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.......

Acknowledgements

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).

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Materials

NameCompanyCatalog NumberComments
Fine Tipped Gel Loading TipsAlpha LaboratoriesGL20057SNTL
Halt Protease Inhibitor Cocktail, EDTA-free 100x 5mLThermoFisher Scientific78437
Handheld homogeniser VWR Collection431-0100
iBlot 2 Gel Transfer DeviceThermoFisher ScientificIB21001
iBlot Transfer Stack, PVDF, regular sizeThermoFisher ScientificIB401031
Image Studio LiteLicorN/AFree download from https://www.licor.com/bio/products/software/image_studio_lite/
IRDye 800CW secondary antibodiesLicor--Select appropriate secondary antibody that is specific against host of primary antibody.
Micro BCA Protein Assay KitThermoFisher Scientific23235
Novex Sharp Pre-stained Protein StandardThermoFisher ScientificLC5800
NuPAGE 4-12% Bis-Tris Protein Gels, 1.0 mm, 15-wellThermoFisher ScientificNP0323BOX
NuPAGE LDS Sample Buffer (4X)ThermoFisher ScientificNP0007
NuPAGE MOPS SDS Running Buffer (20X)ThermoFisher ScientificNP0001
Odyssey Blocking BufferLicor927-40000
Purified Mouse anti-SMN (survival motor neuron) monoclonal antibodyBD Transduction Laboratories610646Is used extensively in the SMN/SMA literature and gives consistent results regardsless of lot number
REVERT Total Protein Stain, 250 mL Licor926-11021 
REVERT Wash Solution Licor926-11012 
RIPA Lysis and Extraction BufferThermoFisher Scientific89900
XCell SureLock Mini-CellThermoFisher ScientificEI0001

References

  1. Bertoni, T. A., Perenha-Viana, M. C., Patussi, E. V., Cardoso, R. F., Svidzinski, T. I. Western blotting is an efficient tool for differential diagnosis of paracoccidioidomycosis and pulmonary tuberculosis. Clinical and Vaccine Immunology. 19 (11), 1887-1888 (2012).
  2. Hutchinson, A. B., et al.

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