#Western blot quantification imagej series#

However, whether IB or LB, the sensitivity of detection of protein or glycans is closely related to the choice of solid phase carrier materials in the process of WB 12, 13. The changes in glycosylation determine the interaction between glycosylation and lectins on the surface of cancer cells 11. For example, the number of terminals sialylated glycans increased significantly in various cancers 8, 9, and the level of core fucosylated glycans also rises obviously 10. It has been reported that during carcinogenic transformation in mammals, severe changes in normal glycosylation occurred. LB is commonly used for detection of protein glycosylation based on lectin–glycan interaction 7. IB is widely used to detect and semi-quantify antigens, determine the relative molecular weight of polypeptide chains and the extraction efficiency of target antigens 6. The process can be described simply as first separating natural or denatured proteins by gel electrophoresis, then transferring the isolated protein bands on the gel to the solid phase supporting material, followed by immunoblotting (IB) and lectin blotting (LB), respectively. Since its inception in 1979, Western blotting (WB) has become a routine experimental method for the determination of proteins and their expression levels in molecular biology and proteomics, as well as used to study protein abundance, kinase activity, cellular localization, protein–protein interaction or monitor protein post-translational modification 5. Therefore, the detection of target protein and changes of abnormal glycosylation are extremely vital for the exploration of new biomarkers for certain diseases. In addition, alpha-1-acid glycoprotein (AGP) as a protein can be used as a prognostic marker of lung cancer 3, and a diagnostic marker for laryngeal cancer 4. Human epidermal growth factor receptor 2 (HER2) is an important indicator for molecular typing of breast cancer, and glycoantigen 19-9 (CA19-9) is used as an important indicator to judge the prognosis of pancreatic cancer 2. What’s more, with the development of proteomics and glycobiology, some proteins and glycoproteins in peripheral blood have become routine tests for diagnosing diseases and predicting prognosis in clinic, such as Alpha-fetoprotein (AFP), a core fucosylated protein used in the diagnosis of liver cancer. Glycoproteins play an important role in cell signaling, immune recognition and cell–cell interaction 1. More than half of the proteins in eukaryotic cells are glycoproteins. Glycosylation is a common post-translational modification of proteins. Protein is the basis of life, which is not only the key component of cell, but also the main executor of biological functions. Bound proteins were stained with direct blue-71, and the staining intensity was quantitated by scanning and densitometry. Moreover, we verified the above results by directly incubating proteins having different molecular weights onto PVDF or NC membranes. The antibody re-probed ability of the two membranes was also explored.

#Western blot quantification imagej series#

Here, by incubating proteins which were transferred to PVDF or NC membranes with a series of antibodies and different types of lectins, we investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein. Which membrane of these two is more suitable for WB has not been reported so far. The most common used membranes are polyvinylidene fluoride (PVDF) and nitrocellulose (NC) membranes. The selection of electroblotted membrane is one of the key factors affecting the detection sensitivity of the protein which is transferred from gel to membrane in WB.

Western blotting (WB) is one of the most widely used techniques to identify proteins as well as post translational modifications of proteins.