For Western blot analysis, whole-cell extracts were prepared using 1X RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA) supplemented with 1X protease inhibitor cocktail (Millipore-Sigma, Burlington, MA, USA). Proteins were separated on 4–12% SDS-PAGE gels and transferred to nitrocellulose membranes. Cell lysates were collected following treatment with compounds for the time indicated. Western blotting used antibodies against FOXM1 (Abcam, Cambridge, UK, catalog number 184637; Cell Signaling Technologies, MA, D12D5), and β-actin (Millipore-Sigma, Burlington, MA, USA, A2228) as an internal loading control. Antibodies against the EMT markers were purchased from Cell Signaling Technologies, Danvers, MA, USA (Cat# 9782) and used at dilutions recommended by the manufacturer. Both IRDye 800 CW goat anti-rabbit secondary antibody (LI-COR, Lincoln, NE, USA, Cat# 926-32211) and IRDye 680 CW goat anti-mouse secondary antibody (LI-COR, Cat# 926-68070) were diluted 1:5000 for incubation with the blots. Band intensities were analyzed with LI-COR Odyssey CLx 2.1 software.
Irdye 680 cw goat anti mouse secondary antibody
Manufactured by LI COR
Sourced in United States
The IRDye 680 CW goat anti-mouse secondary antibody is a fluorescently labeled antibody designed for use in western blotting, ELISA, and other immunoassay applications. It binds to mouse primary antibodies and can be detected using near-infrared fluorescence imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 800cw goat anti rabbit secondary antibody, by LI COR (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Chameleon duo markers, by LI COR (2 mentions)
Precision plus dual color markers, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Odyssey clx 2, by LI COR (1 mentions)
Foxm1, by Cell Signaling Technology (1 mentions)
Nupr1, by Proteintech (1 mentions)
Slc7a11, by Proteintech (1 mentions)
Intercept pbs blocking buffer, by LI COR (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Complete mini protease inhibitor cocktail tablet, by Roche (1 mentions)
4 protocols using irdye 680 cw goat anti mouse secondary antibody
1
Western Blot Analysis of EMT Markers
2
Optimized Western Blot Analysis Protocol
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For Western blot analysis, whole-cell extracts were prepared using 1X RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA) supplemented with 1X protease and phosphatase inhibitor cocktail (Millipore Sigma, Burlington, MA, USA). Proteins were separated on 4–12% SDS-PAGE gels and transferred to nitrocellulose membranes. All antibodies were used at a 1:1000 dilution except for β-actin, which used a 1:40,000 dilution with β-actin as an internal loading control in Western blots (see Supplementary Table S1 for detailed information on antibodies). Both IRDye 800 CW goat anti-rabbit secondary antibody (LI-COR, Cat# 926-32211) and IRDye 680 CW goat anti-mouse secondary antibody (LI-COR, Cat# 926-68070) were diluted (1:5000) for incubation with the blots. Band intensities were analyzed with Licor Odyssey Image Studio 5.2 software that avoids saturation, eliminates comparison of multiple exposures, and allows digital analysis of bands of all intensities, with very accurate protein quantification over a broad linear range. All blots shown together were derived from the same experiment and were processed in parallel. Full uncropped images of blots are shown as Supplementary Figure S3 . Molecular weight markers were Chameleon Duo markers from Licor (8–260 kDa) or Precision Plus Dual Color Markers from Biorad (37–250 kDa).
3
Western Blot Quantification Protocol
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Whole-cell extracts were prepared using 1 × RIPA lysis buffer (Thermo Fisher, Waltham, MA) supplemented with 1 × protease and phosphatase inhibitor cocktail (Millipore Sigma, Burlington, MA). Proteins were separated on 4-12% SDS-PAGE gels and transferred to nitrocellulose membranes. All antibodies were from Cell Signaling Technology (FOXM1, Cat# 5436; ERα, Cat# 8644; LCN2, Cat# 44,058; GPX4, Cat# 52,455; SLC7A11, Cat# 12,691) except for NUPR1, which was from Proteintech (Cat# 15056-1-AP). All were used at a 1:1000 dilution except for NUPR1, used at 1:500, and β-actin, used at 1:5000 dilution as an internal loading control. IRDye 800 CW goat anti-rabbit secondary antibody (LI-COR, Cat# 926-32211) and IRDye 680 CW goat anti-mouse secondary antibody (LI-COR, Cat# 926-68070) were diluted (1:5000) for incubation with the blots. Band intensities were analyzed with LI-COR Odyssey Image Studio 5.2 software that avoids saturation, eliminates comparison of multiple exposures, and allows digital analysis of bands of all intensities, with accurate protein quantification over a broad linear range. Molecular weight markers were Chameleon Duo markers from LI-COR (8-260 kDa) or Precision Plus Dual Color Markers from BioRad (37-250 kDa).
4
Cellular Fractionation and Protein Analysis
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