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NOTE: This protocol has been optimized using standardized, commercially available kits and reagents in order to increase reproducibility (see&#160;Table of Materials).
1. Preparation of samples
<ol>
	<li>Protein extraction
	<ol>
		<li>Transfer snap-frozen cell or tissue samples from -80 &#176;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...

quantitative western blotting to estimate protein expression levels

Source: Huang, Y. T., et al. <a href="https://app.jove.com/v/59438">Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting.</a> 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.

<img alt="Figure 1" src="/files/ftp_upload/21367/21367_Figure1.jpg" /> 
Figure 1. Western blots showing TPS and Smn protein levels in mouse tissues at two different ages.&#160;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 ...

Quantitative Western Blotting to Estimate Protein Expression Levels

Source: Huang, Y. T., et al. Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western ...

Quantitative western blotting &#8212; a total protein normalization-based technique &#8212; is useful for quantifying target protein expression levels.
To begin, load tissue lysate into the wells of a polyacrylamide gradient gel. The lysate contains the target protein, along with other cellular proteins.
Run the gel at an appropriate voltage for the electrophoretic separation of the sample proteins along the gradient. Upon completion, place the gel on top of the polymeric membrane of a transfer stack.
Apply an electrical current to transfer the separated proteins onto the membrane. Next, place the membrane in a fluorescent total protein stain. The charged dye molecules in the stain bind electrostatically to all the proteins on the membrane.
Add a blocking solution. The proteins in the solution bind to the membrane, thus. preventing the non-specific binding of detection reagents. Add a primary antibody binding only to the target protein. Then, add a secondary antibody &#8212; labeled with a fluorophore &#8212; that attaches to the primary antibody.
Excite the sample with a laser. Record the emitted fluorescence from the dye molecules of the total protein stain and the antibody-bound fluorophores at their corresponding wavelengths.
To calculate the normalized protein loading value, divide the total protein signal of each lane by the highest value. Divide the target protein signal by the corresponding normalized loading value to determine the relative expression of the target protein.

quantitative-western-blotting-to-estimate-protein-expression-levels

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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.

Overview

Protocol

<img title="figure-representative results-58" alt="figure-representative results-58" src="/files/ftp_upload/21367/21367_Figure1.jpg" /> 
Figure 1. Western blots showing TPS and Smn protein levels in mouse tissues at two different ages.&#160;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 ...

Quantitative Western Blotting to Estimate Protein Expression Levels

In This Article

Overview

Protocol

Representative Results