Total protein of GC tissue was extracted using a total protein extraction buffer (Beyotime, China) and the protein concentration was measured using a BCA Protein Assay Kit (Pierce Biotechnology, US), according to the manufacturer's instructions. Samples were separated by 10% SDS-PAGE gel electrophoresis and then transferred to nitrocellulose membrane (Millipore, US). Membranes were blocked with 5% (w/v) nonfat milk (BD, US) in Tris-buffered saline (TBS) and incubated with ITGBL1 antibody (dilution 1:500, Abnova, US) diluted in TBS containing 1% (w/v) bovine serum albumin at 4 ℃ overnight. Bound secondary antibodies were detected by Odessey Imaging System (LI-COR Biosciences, Lincoln, NE).
Odessey imaging system
Manufactured by LI COR
Sourced in United States
The Odyssey Imaging System is a high-performance near-infrared fluorescence imaging instrument designed for a wide range of applications in life science research. The system is capable of detecting and quantifying proteins, nucleic acids, and small molecules in a variety of sample types, including western blots, microarrays, and in-cell assays.
Automatically generated - may contain errors
Lab products found in correlation
Nonfat milk, by BD (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Total protein extraction buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Tpck trypsin, by Merck Group (1 mentions)
Evos xl core cell imaging system, by Thermo Fisher Scientific (1 mentions)
Aec substrate chromogen, by Agilent Technologies (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
3 protocols using odessey imaging system
1
Protein Extraction and Western Blot Analysis
2
Influenza Virus Infection Assay
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H1N1 A/England/195/09 or H3N2 A/Hong Kong/1774/99/IAV at a MOI of 1 was preincubated with or without 100 µg/ml of purified FH for 1 hour at 37°C. Virus was then added to A549 cells and allowed to bind cells for 1 hour at 37°C. Cells were washed to remove unbound viruses, and then incubated at 37°C with 5% CO2 in serum free DMEM containing 1% penicillin/streptomycin (Sigma) and 1 µg/ml TPCK Trypsin (Sigma) for either 4 hours or 8 hours. At the appropriate timepoint, cells were either fixed with ice cold 50:50 methanol: acetone for 5 minutes at room temperature, or lysed by scraping into buffer containing 1% Triton X 100, 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, together with 1% anti-proteases cocktail (Thermo Scientific). Fixed cells were immune stained for viral NP protein using mouse anti-NP (Pirbright Institute) and goat anti-mouse HRP conjugate, followed by development with AEC Substrate Chromogen (DAKO); images were acquired via EVOS XL Core Cell Imaging system (Invitrogen). Lysed cells were run on a 10% v/v SDS-PAGE under reducing conditions and western blotting was carried out. The blot was probed using mouse monoclonal anti-M1 (Bio-Rad) and goat anti-mouse IRDye 800CW (LiCor), and visualized using the Odessey Imaging system (LiCor).
3
BG's Effects on PI3K/AKT/GSK-3β Signaling
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Human BCPAP cells were exposed to BG (control, 10 μM/mL and 15 μM/mL) and cultured for 1 day. Using an ice-cold lysis solution containing protease inhibitors, cell lysates were formed for western blotting, and the protein content was measured using a Protein BCA Assay Kit (Pierce Chemical Co., Rockford, USA). The proteins were electrophoretically scattered for a brief period and transferred to a polyvinyl fluoride (PVDF) film, which was blocked and then probed overnight at 4°C using 1:1,000 dilutions of P13K, AKT, GSK-3β, and β-actin primary antibodies. Secondary antibodies (1:5,000) were then added, and the LI-COR Odessey imaging system (Lincoln, USA) was used to stain and visualize the bands to detect the presence of proteins. Densitometry was used to evaluate and quantify the protein band using ImageJ.
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