Tissue lysates were prepared using TissueLyser II (QIAGEN, Valencia, CA, USA) with T-PER Tissue Protein Extraction Reagent and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Total protein concentration was measured by BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA), and equal protein concentrations were resolved by NuPAGE 4%–12% Bis-Tris SDS-PAGE (Thermo Fisher Scientific, Waltham, MA, USA). Electrophoresed proteins were transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Thermo Fisher Scientific, Waltham, MA, USA) and blocked with Odyssey Blocking Buffer (PBS) (Li-Cor Biosciences, Lincoln, NE, USA). Membranes were then incubated with rabbit anti-GO, rabbit anti-LDHA (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HYPDH (Abcam, Cambridge, MA, USA), or rabbit anti-AGT (Abcam, Cambridge, MA, USA) and with mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibody (Abcam, Cambridge, MA, USA). Anti-rabbit IRDye 680 and anti-mouse IRDye 800 secondary antibodies (Li-Cor Biosciences, Lincoln, NE, USA) were used for detection, and signal intensity was measured using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).
Anti mouse irdye 800 secondary antibodies
Manufactured by LI COR
Sourced in United States
The Anti-mouse IRDye 800 secondary antibodies are designed for use in fluorescence-based detection assays. These antibodies are specifically targeted against mouse primary antibodies and are conjugated with the IRDye 800 fluorescent dye. The IRDye 800 has an excitation and emission wavelength that is compatible with the 800 nm channel of LI-COR imaging systems, allowing for sensitive and quantitative detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (4 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Odyssey infrared imaging system, by LI COR (3 mentions)
Nupage 4 12 bis tris sds page, by Thermo Fisher Scientific (2 mentions)
Mouse anti glyceraldehyde 3 phosphate dehydrogenase antibody, by Abcam (2 mentions)
Anti rabbit irdye 680, by LI COR (2 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Tissuelyser 2, by Qiagen (2 mentions)
Odyssey blocking buffer pbs, by LI COR (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Phosphatase inhibitor mix 1, by Serva Electrophoresis (1 mentions)
Protease inhibitor mix g, by Serva Electrophoresis (1 mentions)
3 protocols using anti mouse irdye 800 secondary antibodies
1
Western Blot Analysis of Metabolic Enzymes
2
Western Blotting for Glycogen Synthase
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Tissue lysates were prepared using TissueLyser II (QIAGEN, Valencia, CA) with T-PER Tissue Protein Extraction Reagent and protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Total protein concentration was measured by BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA), and equal protein concentrations were resolved by NuPAGE 4%–12% Bis-Tris SDS-PAGE (Thermo Fisher Scientific, Waltham, MA). Electrophoresed proteins were transferred to nitrocellulose membranes using the iBlot Dry Blotting System (Thermo Fisher Scientific, Waltham, MA) and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences, Lincoln, NE). Membranes were then incubated with rabbit anti-glycogen synthase antibody (Cell Signaling Technology, Danvers, MA) and with mouse anti-glyceraldehyde 3-phosphate dehydrogenase antibody (Abcam, Cambridge, MA). Anti-rabbit IRDye 680 and anti-mouse IRDye 800 secondary antibodies (Li-Cor Biosciences, Lincoln, NE) were used for detection, and signal intensity was measured using the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE).
3
Western Blot Analysis of MAPK and AKT Signaling
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Cells grown at 50% confluency were lysed in RIPA buffer which was supplemented with phosphatase and protease inhibitors (Protease-Inhibitor Mix G and Phosphatase-Inhibitor-Mix I, Serva, Heidelberg, Germany). The cell lysates were clarified by centrifugation, separated by SDS-PAGE, blotted to 0.45 μm nitrocellulose membranes, blocked in 5% nonfat milk in PBS, and probed with primary antibodies. Fluorescent labeled anti-rabbit IRDye 700 and anti-mouse IRDye 800 secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) were used for visualization using an Odyssey infrared imaging system (LI-COR Biosciences). The rabbit phospho-p44/42 MAPK (Erk1/2), rabbit phospho-Akt (Ser473), mouse p44/42 MAPK (Erk1/2), and rabbit AKT primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). The mouse p120RasGAP antibody was obtained from ECM Biosciences LLC (Versailles, KY, USA).
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