![]() ![]() Western blotting is the most widely used method for specific protein detection. Therefore, the characterisation of proteins, including glycan profiling, is crucial for biomarker research, and may provide valuable insights into the disease mechanisms and pathogenesis. Many routine clinical tests utilise the levels of circulating proteins and glycoproteins for diagnostic or prognostic purposes, including the analyses of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in liver function 4, and the pancreatic cancer biomarkers, carcinoembryonic antigen (CEA) and CA 19-9 5. Alterations in the expression and glycosylation of serum proteins can provide information regarding disease stage and malignancy. In eukaryotes, approximately half of all proteins are glycosylated 3. Additionally, it may help the identification of novel diagnostic markers for prostate cancer.Ĭellular proteome is involved in the vast majority of biological functions 1, and proteins play an integral role as the direct executors and regulators of these functions 2. 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. 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. 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. 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. Western blotting is the most extensively used technique for the identification and characterisation of proteins and their expression levels. ![]()
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