Cells were lysed in AB lysis buffer and Western blot analysis was performed. Blots were visualized and bands quantified on an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA) as described(61 (link)). Primary antibodies used were ZEB1 (H-102, Santa Cruz, used at a 1:200 dilution), SEC23A (NBP1-32773, Novus Biologicals, Littleton, CO, USA, used at a 1:5000 dilution), CDH1 (36/E-cadherin, BD Biosciences, used at a 1:5000 dilution), FN1 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA, used at a 1:100 or 1:200 dilution), SERPINE2 (8C4.1, Millipore, Temecula, CA, USA, used at 1:1,000), LRP1 (EPR3724, Abcam, used at 1:5,000), GAPDH (6C5, Thermo Fisher, used at 1:10,000) and ACTB (AC-15, Sigma-Aldrich, St. Louis, MO, USA, used at a 1:10,000 dilution). Secondary antibodies were Anti-Rabbit (Alexa Fluor 680), Anti-Rabbit (Alexa Fluor 800), Anti-Goat (Alexa Fluor 680), Anti-Mouse (Alexa Fluor 680), or Anti-Mouse (Alexa Fluor 800) (Molecular Probes, Eugene, OR, USA), used at a 1:10,000 dilution.
Gapdh 6c5
Manufactured by Thermo Fisher Scientific
Sourced in United States
GAPDH (6C5) is a primary antibody used for the detection of the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein. GAPDH is a key enzyme involved in the glycolytic pathway and is commonly used as a loading control or housekeeping gene in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (2 mentions)
Zeb1 h 102, by Santa Cruz Biotechnology (2 mentions)
Sec23a nbp1 32773, by Novus Biologicals (2 mentions)
Fn1 c 20, by Santa Cruz Biotechnology (2 mentions)
Serpine2 8c4, by Merck Group (2 mentions)
Anti mouse alexa fluor 800, by Thermo Fisher Scientific (2 mentions)
36 e cadherin, by BD (1 mentions)
Mcl 1 sc 819, by Santa Cruz Biotechnology (1 mentions)
Bcl xl sc 8392, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence detection, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Chemidoc digital imaging system, by Bio-Rad (1 mentions)
α flag m2 hrp, by Merck Group (1 mentions)
Image lab package, by Bio-Rad (1 mentions)
Hsc70, by Santa Cruz Biotechnology (1 mentions)
Tsg101, by Merck Group (1 mentions)
Hpa006161, by Merck Group (1 mentions)
5 protocols using gapdh 6c5
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of EMT Markers
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Cells were lysed in AB lysis buffer and western blot analysis was performed. Blots were visualized and bands quantified on an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE, USA) as described.61 (link) Primary antibodies used were ZEB1 (H-102, Santa Cruz Biotechnology, Santa Cruz, CA, USA, used at a 1:200 dilution), SEC23A (NBP1-32773, Novus Biologicals, Littleton, CO, USA, used at a 1:5000 dilution), CDH1 (36/E-cadherin, BD Biosciences, used at a 1:5000 dilution), FN1 (C-20, Santa Cruz Biotechnology, used at a 1:100 or 1:200 dilution), SERPINE2 (8C4.1, Millipore, used at 1:1000), LRP1 (EPR3724, Abcam, Cambridge, UK, used at 1:5000), GAPDH (6C5, Thermo Fisher, used at 1:10 000) and ACTB (AC-15, Sigma-Aldrich, St. Louis, MO, USA, used at a 1:10 000 dilution). Secondary antibodies were anti-Rabbit (Alexa Fluor 680), anti-Rabbit (Alexa Fluor 800), anti-Goat (Alexa Fluor 680), anti-Mouse (Alexa Fluor 680) or anti-Mouse (Alexa Fluor 800) (Molecular Probes, Eugene, OR, USA), used at a 1:10 000 dilution.
3
Western Blot Analysis of Leukemia Cells
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Mouse leukemia cells isolated from the spleen of sick animals were lysed with RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% Triton, 0.5% deoxycholate, and 0.1% SDS), supplemented with a protease inhibitor (Roche). Samples were run on 4–12% Bis-Tris gel (Invitrogen), transferred to PVDF membrane (NOVEX). The membrane was incubated with the primary antibody overnight, followed by 1-hour incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature, washed and incubated with chemiluminescence (ECL) reagent (Thermo Fisher Scientific) and then exposed to film. Densitometric analysis was performed using Image J software (National Institutes of Health). The relative protein level was normalized to the corresponding GAPDH protein level. The following primary antibodies were used for western blotting: Bcl-xl (sc-8392, Santa Cruz Biotechnology), TRAF-1 (sc-6253, Santa Cruz Biotechnology), Mcl-1 (sc-819, Santa Cruz Biotechnology), GAPDH (6C5, Ambion).
4
Quantitative Analysis of MeV Proteins
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BSR-T7/5 cells were transfected in a 12-well plate format (4 × 105 per well) with 2 μg of N-encoding plasmid DNA, and 40 hours after transfection, cells were washed once with phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay buffer [1% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 50 mM tris-Cl (pH 7.2), 10 mM EDTA, 50 mM NaF, 0.05% SDS, protease inhibitors (Roche), 1 mM phenylmethylsulfonyl fluoride]. Cleared lysates (20,000g, 10 min, 4°C) were mixed with 5× urea buffer [200 mM tris (pH 6.8), 8 M urea, 5% SDS, 0.1 mM EDTA, 0.03% bromphenol blue, 1.5% dithiothreitol]. Samples were denatured for 30 min at 50°C, fractionated on 8% SDS–polyacrylamide gel electrophoresis (PAGE) gels, blotted on polyvinylidene difluoride membranes (Millipore), and subjected to enhanced chemiluminescence detection (Pierce) using specific antibodies directed against MeV-N (83KKKII, Millipore), MeV-P (9H4, Abcam), GAPDH (6C5, Ambion), α-FLAG-M2-HRP (Sigma-Aldrich), or HA (16B12, Sigma-Aldrich), as specified. Immunoblots were developed using a ChemiDoc digital imaging system (Bio-Rad). Only nonsaturated images were used for densitometry and carried out using the Image Lab package (Bio-Rad) and global background correction.
5
Immunoblotting and Immunofluorescence Protocol
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Antibodies were used against Calnexin, 1:1000, (WB, rabbit), Dallas, Texas, US; CD81 1:1000 (1.3.3.22, WB, mouse (DLN-09707), Dianova, Hamburg, Germany; EEA1, 1:300 (IF, mouse (610456), BD, New Jersey, NJ, USA; GAPDH (6C5), 1:5000 [WB; mouse (AM4300)], Ambion, Austin, TX, USA; Hsc70 1:2000 (WB; mouse (sc-7298), Santa Cruz, CA, CA, USA; TSG101, 1:1000, (WB, rabbit (HPA006161), Sigma, MO, USA; Wnt3A, 1:500 (WB, rabbit), Abcam, Cambridge, UK, and Ykt6 WB and IF; mouse (sc-365732), Santa Cruz, CA, USA. Antibodies against Ykt6 were generated by immunizing two guinea pigs with the peptides KVSADQWPNGTEATI (aa 105–119, within Longin domain) and YQNPVEADPLTKMQN (aa 131–145, covers part of the SNARE domain). Final bleeds were pooled and affinity purified against the original peptides (Eurogentec). Secondary antibodies directed against the species of interest were coupled to Alexa Fluor 488, 568, 594 and 647 IF, 1:500, Invitrogen, Carlsbad, CA, USA and 680RD and 800CW WB, 1:20,000, LiCor, Lincoln, NE, USA.
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