The expression of PVES/mRNA vaccine was verified by Western blot analysis. Briefly, HEK-293T cells (1 × 106 cells/well) were seeded into a 6-well plate and incubated in a 5% CO2 incubator at 37 °C for 24 h. The PVES/mRNA vaccine complexes at N/P ratio of 32 (5.0 μg mRNA/well) were transfected according to the previous transfection method. After 24 h, the cells were collected, and radio immunoprecipitation assay (RIPA) lysate and proteinase inhibitor were used to lyse the cells. The total protein of the cells was extracted, and a bicinchoninic acid assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration. The same amount of protein was taken for SDS-polyacrylamide gel electrophoresis separation, transferred on to the PVDF membrane, blocked with 5% skimmed milk, detected with SARS-CoV-2 RBD rabbit PAb (1:1000) (Sino Biological, Beijing, China) and secondary antibody (Sino Biological, Beijing, China). The blots were visualized with Clarity Western ECL Substrate (Applygen, Beijing, China) on Chemiluminescence imaging system (Tanon-5200 Multi, Shanghai, China).
Clarity western ecl substrate
Manufactured by Applygen
Sourced in China
Clarity Western ECL Substrate is a chemiluminescent substrate used for the detection of proteins in western blot analysis. It produces a luminescent signal upon reaction with the enzyme horseradish peroxidase (HRP), which is commonly conjugated to secondary antibodies in western blotting protocols.
Automatically generated - may contain errors
2 protocols using clarity western ecl substrate
1
SARS-CoV-2 RBD Protein Expression Verification
2
Comprehensive Analytical Techniques for Biomolecular Characterization
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High-resolution mass spectra (HRMS) were obtained on a Thermo Exactive Plus spectrometer. NMR spectra were recorded on Varian Mercury 400 MHz spectrometers. The purity of final products was determined by high-performance liquid chromatography (HPLC). The UV-B light source (NanJing Nationol Electronic Co. Ltd., China) was a large area irradiation ultraviolet lamp that emitted 106 μW/cm2 at the distance of 10 cm. The wavelength range of UV-B was 280–320 nm, with an average of 302 nm. UV absorption spectra were recorded on a UV-VIS spectrophotometer (UV 1800 SPC, Macy, China) using quartz cuvettes of 1 cm path length. The flow cytometry analyses were performed on a BD FACSCantoTM flow cytometer (λex 488 nm and λem 500–600 nm for phospho-histone H2A.X (Ser139) (20E3) rabbit mAb). Confocal microscopic images were obtained on a Zeiss 780 using the following filters: λex 488 nm and λem 500–600 nm for DCF; λex 488 nm and λem 500–530 nm for Calcein AM; λex 561 nm and λem 600–700 nm for PI. The blots were visualized with Clarity Western ECL Substrate (Applygen, China) on Chemiluminescence imaging system (Tanon-5200 Multi, China).
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