A fixation method for the optimisation of western blotting

Western blotting is the most extensively used technique for the identification and characterisation of proteins and their expression levels. One of the major issues with this technique is the loss of proteins from the blotted membrane during the incubation and washing steps, which affects its sensitivity and reproducibility. Here, we have optimised the fixation conditions for immunoblotting and lectin blotting on electroblotted polyvinylidene difluoride and nitrocellulose membranes, using a combination of organic solvents and heating. Loss of proteins from polyvinylidene difluoride membranes was greatly reduced using this approach, the intensity of lectin blotting and immunoblotting was shown to increase 2.8- to 15-fold and 1.8- to 16-fold, respectively, compared with that samples without treated. Using the optimised method, cystic fibrosis transmembrane regulator and hypoxia-inducible factor 1, two difficult-to-analyse proteins with important physiological and pathological roles, were effectively detected. Additionally, it may help the identification of novel diagnostic markers for prostate cancer.