A fixation method for the optimisation of western blotting

Abstract

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.

High levels of fucosylation and sialylation of milk N‐glycans from mothers with gestational diabetes mellitus alter the offspring gut microbiome and immune balance in mice

“…One gel was stained with Coomassie Brilliant Blue (CBB) R-250, while proteins on another gel were transferred to a 0.45 μm of polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) that were fixed according to our recently developed method. 28 Briefly, the electroblotted PVDF membranes were immersed in acetone at room temperature for 30 minutes with gentle shaking, and followed by sample heating at 100°C for 30 minutes. The fixed membranes were briefly immersed in methanol and then in TBS-T (Tris-buffered saline containing 0.05% Tween-20) for several minutes before their blocking for 1 hour with 5% bovine serum albumin (BSA) in TBS-T. After washing with TBS-T (5 minutes, three times), the membranes were incubated with biotinylated lectins Aleuria aurantia lectin (AAL), Lens culinaris lectin (LCA), Phaseolus vulgaris Erythroagglutinin (PHA-E), and Sambucus nigra lectin (SNA), all diluted 1:20 000 in TBS-T, then at 4°C overnight.…”

“…To enhance the sensitivity of N-glycan analyzed by mass spectrometry, permethylation of N-glycans were performed in a super-clean bench of the temperature-controlled (20°C) laboratory according to the method described previously. 28 Dimethyl sulfoxide (DMSO) containing 1% v/v of distilled water (50 μL) was added to the lyophilized glycan sample under alkaline conditions with powdered sodium hydroxide. Methyl iodide (50 μL) was then added to the mixture, and the reaction was performed at room temperature for 30 minutes with vigorous shaking.…”

Inactivation of Horseradish Peroxidase by Acid for Sequential Chemiluminescent Western Blot

“…Intentionally, we included the YAP (63.7) antibody, which completely loses the reactivity with H 2 O 2 ‐treated blots, and antibodies specific to phosphorylated epitopes including p‐YAP (S127), p‐FAK (Y397), and p‐Erk1/2 (T202/Y204). Of note, the blots were not dried after protein transfer, as drying can fix proteins better on blots . Compared to treatment with H 2 O, treatment with 10% AA (37 °C, 30 min) did not show a consistent effect on epitope recognition by any of the tested antibodies (Figure A,B), suggesting that acid treatment sufficient for complete HRP inactivation causes neither protein loss nor epitope damage.…”

Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current

“…The nitrocellulose membrane is a popular matrix that is frequently used due to its high protein-binding affinity with a pore size of 0.25-0.45 µm in paper-based diagnostics. Protein molecules usually bind to the nitrocellulose membranes through hydrophobic interactions 39 . Due to the ease of their handling, cheap cost, and the presence of hydrophobic interactions between them and the suspended proteins, we tested whether the binding between the SARS-CoV-2 spike protein and antibody could be detected optically when both were added to each other on the nitrocellulose membrane.…”