The rVSVΔG-Sdel-eGFP, rVSV-eGFP (negative control), and rVSVΔG-WH01-eGFP (positive control) were all mixed with protein loading buffers (CWBIO, Jiangsu, China), and the samples were boiled for 10 min. The denatured protein was separated using 10% SDS-PAGE gel electrophoresis (Beyotime), and the proteins were then wet-transferred to the nitrocellulose filter membrane (Beyotime) at 300 mA for 70 min. An amount of 50 g/L skim milk was incubated for 2 h at room temperature. Rabbit anti-SARS-CoV-2 S polyclonal primary antibody was diluted 1:3000 with 30 g/L skim milk and incubated at room temperature for 1.5 h. After five washes with PBST, HRP-conjugated goat anti-rabbit IgG was incubated with the membrane at room temperature for 1 h. Finally, the membrane was washed five times with PBST solution (phosphate-buffered solution containing 0.05% Tween-20) and examined with a Tanon 5200 chemiluminescence imaging system after the addition of electrochemiluminescence (ECL) immunoblotting substrate.
Nitrocellulose filter membrane
Manufactured by Beyotime
Sourced in China
Nitrocellulose filter membrane is a porous filter material made from nitrocellulose. It is commonly used for filtration and separation applications in various industries, such as life sciences, biotechnology, and analytical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Roche (1 mentions)
Mmp13, by Wuhan Servicebio Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Gradient sodium dodecyl sulfate polyacrylamide gels, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemiluminescence system kit, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Ecl detection reagent, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
4 protocols using nitrocellulose filter membrane
1
Western Blot Analysis of SARS-CoV-2 Spike Protein
2
Protein Extraction and Western Blot Analysis
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The cells were lysed using radioimmunoprecipitation assay buffer (RIPA) lysis buffer containing protease inhibitor cocktail (Roche) and phosphatase inhibitors (Roche) on ice for 15 min, the insoluble material was separated by centrifugation (12,000 rpm, 10 min, 4 °C, Centrifuge, Eppendorf, China, 5418R), and the supernatants were collected. The protein concentration was determined by bicinchoninic acid protein (Thermo Fisher Scientific). Equal amounts of protein were subjected to electrophoresis on 10% or 15% gradient sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose filter membrane (Beyotime). The membranes were blocked with 5% BSA (Beyotime) and incubated with specific antibodies, including p-AKT antibody (CST, USA, 9271, 1:1000), AKT antibody (CST, USA, 9272, 1:1000), GAPDH antibody (CST, USA, 2118, 1:10,000), p-mTOR antibody (CST, USA, 2983, 1:1000), mTOR antibody (CST, USA, 2971, 1:1000), LC3 antibody (proteintech, 14600-1-AP, 1:1000), ACAN (Proteintech, 13880-1-AP, 1:500), MMP13 (Servicebio, GB11247-1, 1:1000), SQSTM1 (Abmart, T59081XS, 1:1000), BECN1 (Abmart, T55092XS, 1:1000), GAPDH (Cell Signaling Technology; 1:10,000), and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Licor 926-32211/926-32210).
3
Hippocampal RNA Expression Analysis
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The hippocampi of WT or APP/PS1 mice at age of 11 months were freshly frozen in liquid nitrogen,respectively. The cultured primary mouse astrocytes were plated into 6well plate until ~50% confluency and transfected with mouse Dicer 1 siRNA for 48 h.
Extraction of total RNAs from hippocampi or transfected cells was performed by a kit (TIGEN BIOTECH,Co,LTD.,cat# DP424,China). The isolated RNAs were loaded, separated and transferred onto nitrocellulose filter membrane(Beyotime,cat#FFN02, China) following by UV crosslink. All procedures were performed according to the instruction from Northern TM Max kit(ThermoFisher,cat#AM1940). Following wash,the membrane was incubated with 3'-biotinylaion probes(Stargene Co.,Ltd, Wuhan,China).
The following synthesis 3'-end biotin-probes were used:B2 Mm2 RNA probe,5'-AGCACTGACTACTCTTCTATAAGTC-3'. Mouse U6RNA probe,5'-CAGCACAAAAGGAAACTCACC-3'. The membrane was incubated with secondary streptavidin-HRP antibody(Beyotime,1:800,cat# A0303). The membranes were developed with chemiluminescence system kit(Thermo Fisher).
Extraction of total RNAs from hippocampi or transfected cells was performed by a kit (TIGEN BIOTECH,Co,LTD.,cat# DP424,China). The isolated RNAs were loaded, separated and transferred onto nitrocellulose filter membrane(Beyotime,cat#FFN02, China) following by UV crosslink. All procedures were performed according to the instruction from Northern TM Max kit(ThermoFisher,cat#AM1940). Following wash,the membrane was incubated with 3'-biotinylaion probes(Stargene Co.,Ltd, Wuhan,China).
The following synthesis 3'-end biotin-probes were used:B2 Mm2 RNA probe,5'-AGCACTGACTACTCTTCTATAAGTC-3'. Mouse U6RNA probe,5'-CAGCACAAAAGGAAACTCACC-3'. The membrane was incubated with secondary streptavidin-HRP antibody(Beyotime,1:800,cat# A0303). The membranes were developed with chemiluminescence system kit(Thermo Fisher).
4
Quantitative Immunoblotting of Gastric Cell Signaling
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SGC7901 and BGC723 gastric cells were lysed by RIPA buffer (Beyotime), and protein concentrations were measured by a BCA protein assay kit (Solarbio). Total protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose filter membrane (Beyotime). The membranes were blocked and incubated with BCL2 antibody (No. 15071 1:1000; CST), STAT3 antibody (No. 9139 1:1000; CST), and p-STAT3 antibody (No. 9145 1:1000; CST) followed by incubation with a secondary antibody (1:3000; Solarbio). The protein bands were detected with ECL detection reagent (Thermo Fisher Scientific), and the images were quantified using a Tanon 4600SF system (Tanon, China).
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