Cells were harvested 72h after treatment and centrifuged at 500g for 5 min. media and trypsin was eluted and cells were resuspended in 50–100ul of RIPA buffer. A Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA) was used according to manufacturer’s protocol to quantitate total protein in each sample. 20µg of protein sample was resolved using a SDS-page 15% acrylamide-bis gel and transferred to a nitrocellulose membrane (Advansta, San Jose, CA). Membrane was immunostained using rabbit polyclonal anti-survivin (NB500-201, NOVUS Biologicals, 1:1000) for detection of all survivin isoforms, mouse monoclonal anti-p53 (1:1000) (DO-1, sc126, Santa Cruz Biotech), mouse monoclonal anti-YY1 (1:1000) (H-10, sc-7341 Santa Cruz Biotech), rabbit polyclonal anti-sp1 (1:1000) (PEP-2 sc59, Santa Cruz Biotech) and loading controls rabbit monoclonal anti-β-actin (1:5000) (D6A8, Cell Signaling) and mouse monoclonal anti-GAPDH (1:1000) (O411, sc-47724, Santa Cruz Biotech). Dylight 800 Goat-anti-mouse IGG (1:20000) (SA535521, Invitrogen), and goat-anti-rabbit IGG antibodies (1:20000) (SA5-35571, Thermo Scientific) were used for secondary stain and quantitation via Odessey CL-x (LI-COR Lincoln, NE).
Nitrocellulose membrane
Manufactured by Advansta
Sourced in United States
Nitrocellulose membrane is a laboratory material used for protein and nucleic acid blotting techniques. It is a thin, porous sheet made from nitrated cotton fibers that allows the transfer and immobilization of biomolecules for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Odessey clx, by LI COR (1 mentions)
Sa5 35571, by Thermo Fisher Scientific (1 mentions)
Nb500 201, by Novus Biologicals (1 mentions)
Sa5 35521, by Thermo Fisher Scientific (1 mentions)
Ab15348, by Abcam (1 mentions)
Ab92323, by Abcam (1 mentions)
Ab49900, by Abcam (1 mentions)
Complete mini edta free protease inhibitor, by Merck Group (1 mentions)
Hpa000288, by Merck Group (1 mentions)
Eblot l1, by GenScript (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Fusion fx7 imaging system, by Vilber (1 mentions)
Westernbright quantum, by Advansta (1 mentions)
Mouse anti β actin, by Proteintech (1 mentions)
Donkey anti goat igg, by Jackson ImmunoResearch (1 mentions)
Goat anti emmprin, by R&D Systems (1 mentions)
Goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
3 protocols using nitrocellulose membrane
1
Quantitative Protein Analysis of Cellular Stress Pathways
2
Protein Expression and Quantification
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Cells were lysed using RIPA buffer (Thermo Fisher Scientific) containing cOmplete, Mini, EDTA-free protease inhibitor (Sigma-Aldrich). Protein concentration was determined by BCA Protein Assay kit (Thermo Fisher Scientific). 10 µg/sample with 4x Laemmli Buffer (Bio-Rad, Hercules, CA, USA) containing β-mercaptoethanol was resolved in 20% Mini-PROTEAN TGX Gel (Witec AG, Sursee, Switzerland). PNGase treatments have been performed according to the manufacturer’s protocol (New England BioLabs, Frankfurt, Germany). Gels were transblotted on nitrocellulose membrane (Advansta) using eBlot L1 (GenScript, Piscataway, NJ, USA). Membranes were blocked and stained with primary antibody overnight in 5% nonfat dry milk in 0.1% phosphate-buffered saline Tween, visualized with horseradish peroxidase (HRP)-conjugated secondary antibody using WesternBright Quantum (Advansta Corporation, Menlo Park, CA, USA) and imaged by Fusion FX 7 Imaging System (Vilber Lourmat, Eberhardzell, Germany). Antibodies used are rabbit anti-ACE2 (1:500, ab15348, Abcam, Cambridge, UK), rabbit anti-ACE2 (1:1000, HPA000288, Sigma-Aldrich), rabbit anti-TMPRSS2 (1:1000, ab92323, Abcam), rabbit anti-NRP1 (1:50, HPA030278, Sigma-Aldrich), HRP-conjugated anti-rabbit (1:10000, 11-035-003, Jackson), and HRP-conjugated anti β-actin (1:25000, ab49900, Abcam).
3
Western Blot Quantification of Protein Levels
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Equal amounts of cellular lysates (20 μg/lane) that were extracted in RIPA buffer were loaded on a gradient 4-20% SDS-PAGE, and proteins were separated by electrophoresis and transferred onto a nitrocellulose membrane (Advansta, San Jose, CA, USA). Block-Chemi (Advansta) reagent served to block the membranes for 1 hour. Then the primary antibodies goat anti-EMMPRIN (R&D systems, diluted 1:1000), mouse anti-TRAF6 antibody (Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:500) or mouse anti-β-actin (ProteinTech, Rosemont, IL, USA, diluted 1:10,000) in blocking solution were added for overnight incubation at 4°C, following 3 washes in TBST buffer (1xTris-buffered saline with 0.1% Tween 20). Then the HRP-conjugated secondary antibodies donkey anti-goat IgG or goat-anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, USA, diluted 1:5,000) was incubated for 1 hour, and after three more washes, the membranes were incubated with the WesternBright ECL HRP substrate (Advansta). The protein bands were visualized using the Omega Lum G imaging system (Aplegen, Pleasanton, CA, USA).
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