Quantitative Western Blotting to Estimate Protein Expression Levels

Abstract

Source: Huang, Y. T., et al. Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting. J. Vis. Exp. (2019).

The video demonstrates a total protein normalization-based western blotting technique for quantifying the target protein expression in a sample. By utilizing the total protein staining of the sample, a ratiometric analysis is performed to determine the relative expression level of the target protein.

Protocol

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

1. Protein extraction
   1. Transfer snap-frozen cell or tissue samples from -80 °C on dry ice, thaw on ice, and wash as required with ice-cold 1x phosphate-buffered saline (PBS). Avoid unnecessary freeze-thaw cycles as this will affect protein quality.
   2. Add radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0....

Results

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-la.......

Materials

List of materials used in this article

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 regardless 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