Proteins were extracted using cell lysis buffer RIPA (Beyotime, Shanghai, China) and a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). A total of 20 μg protein/per lane was separated by 4–20% SDS-PAGE gels and subsequently transferred onto a membrane and blocked with 5% skim milk (Difco; BD Biosciences) at room temperature for 2 h. The membranes were then subjected to immunoblotting with appropriate primary at 4°C overnight and secondary antibodies for 2 h at room temperature. Luminescence was visualized using a BeyoECL Moon super sensitivity detection kit (Beyotime, Shanghai, China) according to the manufacturer's protocol.
Beyoecl moon super sensitivity detection kit
Manufactured by Beyotime
Sourced in China
The BeyoECL Moon super sensitivity detection kit is a lab equipment product designed for highly sensitive detection applications. It provides a core function of enabling enhanced chemiluminescence-based detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Beyotime (3 mentions)
Cell lysis buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa cell lysis buffer, by Beyotime (1 mentions)
Image pro plus v6, by Media Cybernetics (1 mentions)
Rabbit anti nf κb p65, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Mouse anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Rabbit anti phospho nf κb p65 ser536, by Cell Signaling Technology (1 mentions)
4 protocols using beyoecl moon super sensitivity detection kit
1
Protein Extraction and Western Blot Analysis
2
Western Blot Protein Analysis Protocol
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Proteins were extracted using cell lysis buffer (Beyotime, Shanghai, China) and a protease and phosphatase inhibitor cocktail (Beyotime, Shanghai, China), separated by 4-20% SDS-PAGE gels and were then subjected to immunoblotting with appropriate primary and secondary antibodies (Supplementary Table S2 ). Luminescence was visualized using a BeyoECL Moon super sensitivity detection kit (Beyotime, Shanghai, China).
3
Quantitative Western Blot Analysis of Protein Targets
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Total protein was extracted using cell lysis buffer (Beyotime Institute of Biotechnology) supplemented with a protease and phosphatase inhibitor cocktail (Beyotime Institute of Biotechnology). The protein concentration was quantified using a bicinchoninic acid protein estimation kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. A total of 20 µg protein/per lane was loaded on 4-20% SDS-gels and resolved using SDS-PAGE, and subsequently transferred to a PVDF membrane (MilliporeSigma). The membranes were rinsed with Trisbuffered saline with 0.1% Tween-20 (TBST) for 5 min and blocked by 5% skimmed milk powder at room temperature for 2 h. The membranes were then subjected to immunoblotting with the appropriate primary (WBSCR22; cat. no. A7317; 1:1,000; ABclonal Biotech Co. and ISG15; cat. no. P05161; 1:1,000; Cusabio technology LLC.) at 4°C overnight and goat anti-rabbit (HRP) secondary antibodies (cat. no. orb43514; 1:5,000; Biorbyt) for 2 h at room temperature. The GAPDH antibody (cat. no. orb555879; 1:5,000; Biorbyt) was used as a control. Signals were visualized using the BeyoECL Moon super sensitivity detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol, and the blots were quantified using Image Pro Plus v6.0 software (Media Cybernetics, Inc.).
4
Quantitative Analysis of Inflammatory Signaling Proteins
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Protein extraction was performed on BV2 and mouse brain samples using RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with a 1 mM protease inhibitor cocktail (B14001, Selleck, Houston, TX, USA). The lysates were then centrifuged to gather supernatants. Nuclear and cytoplasmic proteins were extracted using a cytoplasmic and nuclear protein extraction kit (AR0106, Boster, Wuhan, China). The proteins were transferred onto a PVDF membrane (Millipore, Shanghai, China) after being resolved using suitable SDS-PAGE. The membrane was blocked in 5% non-fat milk solution for one hour at room temperature, then incubated overnight at 4 °C with primary antibodies. Immunoblotting was identified using horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibodies, in conjunction with the BeyoECL Moon super sensitivity detection kit (Beyotime, Shanghai, China). The antibodies and concentrations used for Western blotting were as follows: rabbit anti-TonEBP (ab3446, Abcam, Waltham, USA) 1:1000, rabbit anti-NF-κB p65 (8242, Cell Signaling Technology, Danvers, MA, USA) 1:2000, rabbit anti-phospho-NF-κB p65 (Ser536) (3033, Cell Signaling Technology, Danvers, USA) 1:500, mouse anti-PELI1 (TA807629S, ORIGENE, Rockville, MD, USA) 1:2000, and mouse anti-GAPDH (60004-1-Ig, Proteintech, Wuhan, China) 1:50,000.
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