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

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.

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

“…In this study, the 9-LOX recombinant protein was recovered from the cell lysates. SDS-PAGE analysis showed a band size of approximately 97KDa of expected band size that was confirmed by immunoblotting of PVDF membrane probed using anti-histidine antibody which is very good for lowly expressed proteins although it gives a high background (Xiang et al ., 2021). The expression results from this study are similar to those from a study by Rodrigo et al (2013) where expressed recombinant protein was recovered from the cell lysates as was revealed by SDS-PAGE and Western blot analysis.…”

Mass Spectrometry versus Conventional Techniques of Protein Detection: Zika Virus NS3 Protease Activity towards Cellular Proteins

“…10–100 pg for colorimetric detection. Examples of detection limit calculations for WB can be found elsewhere [ 48 , 49 , 50 , 51 ]. Mass spectrometry is also seen as a very sensitive analytical technique.…”

Methods of Sputum and Mucus Assessment for Muco-Obstructive Lung Diseases in 2022: Time to “Unplug” from Our Daily Routine!

“…The technique does, however, give no information on mucus composition. The dot-blot assay is also a simple technique for detecting glycoproteins but severely lacks in sensitivity and specificity [ 117 ].…”