PANC-1 or AsPC-1 cells were cultured in six-well plates and total protein was extracted by RIPA lysis buffer (Beyotime, China). After the cell lysates were sonicated, and treated with the BCA Protein Analysis kit (Beyotime, China) to detect the concentration of protein by using an enzyme-labeled instrument (Molecular Devices, USA). Proteins were used to SDS-page and transferred to nitrocellulose filter membrane (Pall Corporation, USA), blocked with rapid blocking buffer (GenScript, USA) for 17 min and incubated with primary antibody overnight at 4 °C. The next day it was washed with TBST and incubated with secondary antibodies for 40 min, the membranes were scanned and analyzed with Odyssey infrared imaging instrument (LI-COR, USA). Antibodies used in this study include: anti-β-actin (1:20,000, ABclonal, China), anti-YAP1 (1:1500, Cell Signaling Technology, USA), anti-CTGF (1:1000, Novus Biologicals, USA), and secondary antibody (800R rabbit antibody, 1:1000, LI-COR, USA).
Odyssey infrared imaging instrument
Manufactured by LI COR
Sourced in United States
The Odyssey infrared imaging instrument is a laboratory equipment designed for the detection and quantification of proteins, nucleic acids, and other biomolecules. It utilizes near-infrared fluorescence technology to generate high-resolution images and accurate data.
Automatically generated - may contain errors
Lab products found in correlation
800cw labeled secondary antibodies, by LI COR (3 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Iblot system, by Thermo Fisher Scientific (3 mentions)
Lysis buffer, by Cell Signaling Technology (2 mentions)
Anti yap1, by Cell Signaling Technology (1 mentions)
Bca protein analysis kit, by Beyotime (1 mentions)
Anti ctgf, by Novus Biologicals (1 mentions)
Nitrocellulose filter membrane, by Pall Corporation (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti β actin, by ABclonal (1 mentions)
Enzyme labeled instrument, by Molecular Devices (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ab125066, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Odyssey 2, by LI COR (1 mentions)
6 protocols using odyssey infrared imaging instrument
1
Protein Expression Analysis of PANC-1 and AsPC-1 Cells
2
Quantitative Protein Expression Analysis
3
Western Blot Analysis of Factor V
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Proteins were subjected to gel electrophoresis using 4% to 12% gradient or 10% NuPage gels (Invitrogen) under reducing conditions using Mops. Proteins were subsequently transferred onto nitrocellulose membranes using a dry iBlot system (Invitrogen) followed by blocking with Roche proprietary blocking reagent. Membranes were probed with a mouse antihuman FV HC mAb (AHV #5146, primary antibody) followed by rabbit antimouse IgG antibody conjugated to IRDyLight 800 (fluorescently labeled secondary antibody). Proteolytic products were visualized by scanning blots in an Odyssey Infrared Imaging Instrument (Li-cor Biosciences).
4
Protein Extraction and Western Blot Analysis
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RIPA buffer (Solarbio, Beijing, China) with proteinase inhibitors was used to isolate proteins from PC cells. The BCA kit (Beyotime) was used to determine the concentration of proteins. 20 μg protein were used for detection. SDS–polyacrylamide gels were applied to separate different proteins. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were cultured with 5% fat-free milk for 2 h at 37 °C. The membranes were then cultured with primary antibodies overnight at 4 °C. Anti-GPX4 (ab125066, 1:2000), anti-beta-catenin (ab32572, 1:1000) were obtained from Abcam (Cambridge, UK). The membranes were washed with PBST for 5 min three times and incubated with the indicated secondary antibodies for 45 min. Proteins were detected using an Odyssey infrared imaging instrument (LI-COR, USA).
5
Protein Quantification and Immunoblot Analysis
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Total protein was isolated using cell lysis buffer (Cell Signaling Technologies, Danvers, MA), as previously described [8 (link)]. Briefly, 40μg of protein per lane was resolved by SDS—PAGE, followed by transfer to nitrocellulose membrane and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE). The membranes were probed with the following primary antibodies, using the indicated dilutions: rabbit anti-HK (1/500), goat anti-PFK (1/1000), rabbit anti-PK (1/1000), rabbit anti-pAMPK (1/500), rabbit anti-pAkt (1/1000), rabbit anti-FOXO1 (1/500), mouse anti-tubulin (1/5000) and rabbit anti-PDK4 (1/500). This was followed by treatment with infrared IR-dye 700CW and/or 800CW-labeled secondary antibodies (Li-Cor Biosciences, Lincoln, NE). Blots were scanned and quantified using Li-Cor Odyssey infrared imaging instrument and Odyssey 2.1 software.
6
Quantitative Protein Expression Analysis
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Total proteins were prepared using lysis buffer (Cell Signaling Technologies, Danvers, MA) and resolved by SDS–PAGE electrophoresis. Proteins were transferred to nitrocellulose membrane using the iBlot system (Life Technologies, Calrsbad, CA) and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE). The membranes were probed with the following primary antibodies, using the indicated dilution: rabbit anti-GK (1/500), goat anti-PFK (1/1000), anti-PEPCK (1/1000), anti FBPase (1/500), rabbit anti-pAMPK (1/500), mouse anti-AMPK (1/1000), rabbit anti-FOXO1 (1/500), mouse anti-tubulin (1/5000), rabbit anti-PDK4 (1/500), mouse anti-PDH (1/1000), rabbit anti-pPDH (1/500), and goat anti-GKRP (1/2000). This was followed by treatment with infrared IR-dye 700CW and/or 800CW-labeled secondary antibodies (Li-Cor Biosciences, Lincoln, NE). The blots were then scanned and protein levels quantified using Li-Cor Odyssey infrared imaging instrument and Odyssey 2.1 software.
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