Cells were lysed in ice-cold buffer containing 50 mmol/L Tris-HCl, pH 7.4, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mmol/L NaCl, 10 mmol/L NaF, 1 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L Pefabloc SC, and 2 mg/mL protease inhibitor cocktail (Roche Diagnostics Corp). Protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad Laboratories). Cell lysates containing 12.5 to 25 μg of protein were analyzed by SDS-PAGE and immunoblotting. Primary antibodies used include the following: Cav-1 (No. 610058; BD Biosciences), LC3B (No. 2775; Cell Signaling), SQSTM1/p62 (No. ab56416; Abcam), and β-actin (No. sc-69879; Santa Cruz Biotechnology), PKB/AKT (protein kinase B, also known as Akt; No. 9272; Cell Signaling), p-AKT (No. 9277; Cell Signaling), pS6K (No. 9205; Cell Signaling), S6K (No. 9202; Cell Signaling), ATG5 (No. 2630; Cell Signaling), connexin-43 (Cx-43; No. ab11370; Abcam), vinculin (No. v9131; Sigma), and HSP90 (No. 610419; BD Biosciences). Secondary antibodies were fluorescence-labeled antibodies (LI-COR Biotechnology). Bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology). Densitometry analysis was performed with ImageJ software (National Institutes of Health) or Image Studio Lite (LI-COR Biosciences).
Fluorescence labeled antibodies
Manufactured by LI COR
Fluorescence-labeled antibodies are lab equipment used for various applications in biological and medical research. They consist of antibodies that have been chemically conjugated with fluorescent dyes. The fluorescent labeling enables the detection and visualization of target molecules, proteins, or cellular structures in samples using fluorescence-based techniques, such as flow cytometry and fluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Dc protein assay kit, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Odyssey infrared system, by LI COR (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Abcam (1 mentions)
Vinculin, by Merck Group (1 mentions)
Image studio lite, by LI COR (1 mentions)
Hsp90, by BD (1 mentions)
Pefabloc, by Roche (1 mentions)
Cav 1, by BD (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
P s6k, by Cell Signaling Technology (1 mentions)
Pkb akt, by Cell Signaling Technology (1 mentions)
Connexin43 cx43, by Abcam (1 mentions)
Pefabloc sc, by Roche (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
4 protocols using fluorescence labeled antibodies
1
Cell Lysis and Protein Analysis
2
Protein Analysis via SDS-PAGE and Immunoblotting
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Cells were lysed on ice with lysis buffer (50 mM Tris·HCl pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mM NaCl, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc SC, and 2 mg/ml protease inhibitor mixture; Roche Diagnostics). Protein concentrations were determined with the DC Protein assay kit (Bio-Rad Laboratories). Lysates were analyzed by SDS/PAGE and immunoblotted. Primary antibodies used include the following: LRP5, LRP6, phospho-β-catenin, Cyclin D1, GR, LC3A/B, P62 and GAPDH as the loading control. Secondary antibodies were fluorescence-labeled antibodies (LI-COR Biotechnology). Bands were visualized with the Odyssey Infrared LI-COR system (LI-COR Biotechnology).
3
Mammalian Cell Lysis and Protein Analysis
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Cells were lysed on ice with lysis buffer (50 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 100 mM NaCl, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc SC, and 2 mg/ml protease inhibitor cocktail (Roche Diagnostics)). Protein concentrations were determined with the DC Protein assay kit (Bio-Rad Laboratories). Lysates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting. Secondary antibodies were fluorescence-labeled antibodies (LI-COR Biotechnology). Bands were visualized with the Odyssey Infrared Licor system (LI-COR Biotechnology).
4
Protein Extraction and Analysis from Snap-Frozen Tissues
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Tissues were snap frozen in liquid nitrogen, pulverized, and resuspended in lysis buffer (50 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 100 mM NaCl, 10 mM NaF, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM Pefabloc SC, and 2 mg/ml protease inhibitor cocktail (Roche Diagnostics)). Cells were lysed on ice with lysis buffer. Protein concentrations were determined with the DC Protein assay kit (Bio-Rad Laboratories). Lysates were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting. Primary antibodies used include the following: iNOS (Cayman) and GAPDH (Affinity Bioreagents). Secondary antibodies were fluorescence-labeled antibodies (LI-COR Biotechnology). Bands were visualized with the Odyssey Infrared Licor system
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