Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Advantages of Stain-Free Technology

Abstract

Western blotting is one of the most used techniques in research laboratories. It is popular because it is an easy way of semiquantifying protein amounts in different samples. In Western blotting, the most commonly used method for controlling the differences in the amount of protein loaded is to independently quantify housekeeping proteins (typically actin, GAPDH or tubulin). Another less commonly used method is total protein normalization using stains, such as Ponceau S or Coomassie Brilliant Blue, which stains all the proteins on the blots. A less commonly used but powerful total protein staining technique is stain-free normalization. The stain-free technology is able to detect total protein in a large linear dynamic range and has the advantage of allowing protein detection on the gel before transblotting. This chapter discusses the theory, advantages, and method used to do total protein quantification using stain-free gels for normalization of Western blots.

Keywords: Immunoblotting; Loading control; Stain-free technology; Total protein normalization; Western blotting.

Fig. 1.

Detection of total mouse heart protein on stain-free gel. (A) Detection of 10–80 μg of mouse heart homogenate using stain-free technology. The Criterion stain-free gel was exposed to 1 min of UV transillumination using the ChemiDoc MP to activate it before imaging. (B) Quantification of the relative intensity of total protein detected on the gel versus the total protein amount loaded onto the gel (μg) (n = 3 for each data point). Data are represented as mean ± standard deviation.

Fig. 2.

Quantification of total mouse heart protein detected on nitrocellulose membrane using stain-free technology. (A) Detection of 10–80 μg of mouse heart homogenate on nitrocellulose membrane using stain-free technology. (B) Quantification of the relative intensity of total protein detected on the membrane versus the total protein amount loaded onto the gel (μg) (n = 3 for each data point). Data are represented as mean ± standard deviation.

Fig. 3.

Quantification and normalization of 20S proteasome β1 levels in mouse heart homogenates. (A) Western blot of mouse heart homogenate (10–80 μg) using an antibody against the β1 subunit of the 20S proteasome. (B) Quantification of 20S proteasome β1 levels (10–80 μg), not normalized and normalized to total protein detected using stain-free technology on the nitrocellulose membrane (n = 3 for each data point). Data are represented as mean ± standard deviation. (C) Quantification of 20S proteasome β1 levels (10–40 μg), not normalized and normalized to total protein detected using stain-free technology on the nitrocellulose membrane (n = 3 for each data point). Data are represented as mean ± standard deviation.