Protein expression / Western blot

Objectives

Western blot allows the determination of the expression of a protein in its native or active form in tissues and helps to better understand the mechanism of action of an active compound.

Summarized methodology

Several steps are to be taken into account in order to optimize the results to be obtained:

Tissue harvesting and homogeneization (proteases and phosphatases inhibitors when necessary).
Protein separation on denaturing SDS-polyacrylamide gel
Primary and secondary antibodies (HRP-linked or biotinylated followed by the addition of streptavidin-HRP) concentrations and conditions of incubation

Figure 1: Representative immunoblots of α-actin in the corpus cavernosum (upper panel) and aorta (lower panel) of WKY and SHR at 6, 12 and 24 wk of age. Equal amounts of protein were loaded on each lane. (from Behr-Roussel et. al 2005).

Endpoints

The proteins of interest are detected by enhanced chemiluminescence and visualized by immediate exposure to autoradiographic film (figure 1). Each sample is assayed in duplicate or triplicate and densitometric results are averaged per animal, and the results are normalized to the relative density of the internal control.
Although the examination of the expression a wide variety of proteins could be performed, the expression of the following proteins has been validated at Pelvipharm :

Related Pelvipharm bibliography

Oudot, A. et al.
J Sex Med (2010) : 7(1)p1:79-88

Behr-Roussel, D. et al.
Am J Physiol – Regul (2005) : 288:R276-R283

Links to applicable Targeted disorders / Pathophysiological models

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