Comparison of the sensitivity of Western blotting between PVDF and NC membranes

Yufang Xiang,Zhi Li,Shaobo Liu,Yuanyuan Zheng,Gang Liu,Weijie Dong

doi:10.1038/s41598-021-91521-8

Abstract

Western blotting (WB) is one of the most widely used techniques to identify proteins as well as post translational modifications of proteins. 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. The most common used membranes are polyvinylidene fluoride (PVDF) and nitrocellulose (NC) membranes. Which membrane of these two is more suitable for WB has not been reported so far. 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. The antibody re-probed ability of the two membranes was also explored. Moreover, we verified the above results by directly incubating proteins having different molecular weights onto PVDF or NC membranes. Bound proteins were stained with direct blue-71, and the staining intensity was quantitated by scanning and densitometry.

Highlights

Protein is the basis of life, which is the key component of cell, and the main executor of biological functions
We investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein, by incubating the proteins which transferred to polyvinylidene fluoride (PVDF) or NC membranes with a series of antibodies and different types of lectins
The results showed that under the same electrophoretic conditions, as for visualization of IgG, PVDF and NC membranes need at least 0.7 μg of serum protein, the gray analysis of staining bands showed that when the amount of protein in sera was between 0.7 and 3.0 μg, compared with PVDF, the amount of protein transferred to NC membrane was significantly increased

Summary

Introduction

Protein is the basis of life, which is the key component of cell, and the main executor of biological functions. Nitrocellulose (NC) and polyvinylidene fluoride (PVDF) membrane are the two solid phase carriers widely used to bind proteins in WB. Clinical samples are often very precious, so sometimes they need to be re-probed Both PVDF and NC membranes have been reported as carrier materials[16,17]. We investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein, by incubating the proteins which transferred to PVDF or NC membranes with a series of antibodies and different types of lectins. The results of this study can provide basic reference data for the selection of solid phase carriers in WB, so as to save experimental raw materials and improve sensitivity, especially for precious clinical samples

Methods

Results

Conclusion