Quantitative Western Blotting to Estimate Protein Expression Levels

Published: May 31, 2023

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 Protein extraction 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 aff…

Representative 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-lane TPS was calculated and is indicated (in arbitrary units). M: marker/protein standard; kDa: kilodalton; a.u.: arbitrary unit; P5: postnatal …

Açıklamalar

The authors have nothing to disclose.

Materials

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

Etiketler

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Bu Makaleden Alıntı Yapın
Quantitative Western Blotting to Estimate Protein Expression Levels. J. Vis. Exp. (Pending Publication), e21367, doi: (2023).

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