Cells were lysed in 62.5 mM Tris pH 6.8, 2% SDS, 10% Glycerol, with protease-inhibitor cocktail (Cell Signaling). Protein concentration was measured using DC Protein Assay (Bio-Rad), and Western blot analysis was performed. The primary antibodies used are listed in Table 1 (in supplement) . Secondary horse radish peroxidase conjugated anti-mouse and anti-rabbit antibodies were from Jackson ImmunoResearch or BioRad. GAPDH (Am4300) (Ambion) was used as an internal control. Antibodies were diluted 1:1000 in TBS/1%BSA/0.05% Tween20 buffer except for GAPDH using a 1:5000 dilution.
Gapdh am4300
Manufactured by Thermo Fisher Scientific
Sourced in United States
GAPDH #AM4300 is a probe designed for the detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression. GAPDH is a housekeeping gene commonly used as an internal control in gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Anti ha peroxidase, by Roche (1 mentions)
Pser36 shc1, by Abcam (1 mentions)
α tubulin, by Merck Group (1 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Ab129002, by Abcam (1 mentions)
Ab40794, by Abcam (1 mentions)
Ab32084, by Abcam (1 mentions)
Ab81298, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Goat anti mouse igg h l hrp, by Thermo Fisher Scientific (1 mentions)
Ibind western system, by Thermo Fisher Scientific (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Chemidoc touch mp system, by Bio-Rad (1 mentions)
G 21040, by Thermo Fisher Scientific (1 mentions)
7 protocols using gapdh am4300
1
Western Blot Analysis of Protein Samples
2
Muscle Protein Immunoblotting Protocol
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Whole quadriceps muscles from both legs were frozen in liquid nitrogen while being pulverized together into a powder. A homogenous sample of pulverized muscle was used for immunoblotting (Drew et al., 2014 ). Proteins from each individual whole‐cell homogenate were normalized (expressed relative to the pixel densitometry) to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH, AM4300; Ambion, Foster City, CA, USA). Phosphorylation‐specific proteins were normalized (expressed relative to pixel densitometry) to the same unphosphorylated protein (i.e., phosphorylated Drp1 at Ser 616 was expressed relative to the pixel densitometry of Drp1 for each individual sample). In many cases, membranes were cut so that multiple proteins could be probed with different antibodies simultaneously. This allows for conservation of sample and reagents. Phosphoprotein blots were stripped and reprobed with antibody against the protein of interest. GAPDH protein or the mitochondrial‐specific HSP60 were assessed for every membrane to ensure equal loading of all lysates. See Table S1 for a list of the primary antibodies used.
3
Protein Isolation and Detection Workflow
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Proteins were isolated and detected as described previously23 (link)24 (link)62 (link). Primary antibodies raised against following proteins were used: phospho-p38 MAPK (9212), phospho-MAPKAP kinase 2 (MK2) (3044), MAPKAP kinase 2 (3042), phospho γH2AX (9718), phospho-ATF2 (9221) from Cell Signaling Technology, Boston, MA), p38MAPKα (sc-535), phospho ERK (sc-16982R), ERK1 (sc-94), JNK (sc-571), ATF2 (sc-187) from Santa Cruz Biotechnology, Santa Cruz, CA), pJNK (AF1205), R&D Systems Minneapolis, MN, USA, GAPDH (AM4300, Ambion, Grand Island, NY), a-tubulin (T5168, Sigma Aldrich, Dorset, UK), Shc1 (610879), BD Biosciences, San Diego, CA, USA, pSer36-Shc1 (54518, Abcam, Cambridge, UK) and anti-HA-Peroxidase (12013819001), Roche, Mannheim, Germany. Antibodies were visualized by ECL Western blot detection reagents (Amersham, Buckinghamshire, UK), quantified by densitometric scanning using the image J program (NIH, Bethesda, MD) and normalized against loading control.
4
Western Blot Analysis of VE-Cadherin and GAPDH
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Protein samples (10 µg in 10 µl) and Precision Plus Blue marker (Bio-Rad) were loaded on 4–15% mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad) for electrophoresis and transferred to a polyvinylidene difluoride membrane using a Trans-blot Turbo Transfer System (Bio-Rad). Non-specific binding was blocked with 5% (w/v) non-fat dry milk (Elk) in a mixture of tris-buffered saline and polysorbate 20 (TBS-T) for 1 h at RT. The membranes were incubated with the primary antibodies in 1% (w/v) milk in TBS-T (Vascular Endothelial (VE)-cadherin #19–3600, 1:500, ThermoFisher Scientific and glyceraldehyde 3-phosphate dehydrogenase (GAPDH #AM4300, 1:5000, ThermoFisher Scientific) overnight at 4 °C. The membranes were washed four times with TBS-T, incubated with the secondary antibodies in 1% (w/v) milk in TBS-T (anti-rabbit IRdye 680 or anti-mouse IRdye 800, respectively, 1:1000) for 1 h at RT and washed 5 times with TBS-T. The membranes were scanned with Odyssey ClX (Li-cor). Finally, western blots were analysed using Fiji to calculate the relative intensity of each band, corrected for GAPDH.
5
Quantifying FA Protein Expression
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The expression levels of FA proteins were quantified using western blotting (WB) at room temperature (RT). Whole cell proteins were extracted using NuPAGE™ LDS Sample Buffer (Thermo Fisher, Waltham, MA, USA), loaded on Bolt™ Bis-Tris Protein Gel (Thermo Fisher), and blotted onto 0.45 μm PVDF membranes (Thermo Fisher). Proteins were quantified using a Qubit™ Protein Broad Range Assay and Qubit 4 Fluorometer (Thermo Fisher) following the manufacturer’s instruction. Membranes were finally incubated with primary antibodies: GAPDH (AM4300, Thermo Fisher), vinculin (ab129002, abcam), paxillin (ab32084, abcam), FAK (ab40794, abcam), and phospho Y397 FAK (ab81298, abcam) and secondary antibodies: Goat Anti-Rabbit IgG H&L HRP (ab205718, abcam) and Goat Anti-Mouse IgG H&L HRP (G21040, Thermo Fisher) using iBind Western System (Thermo Fisher). All membranes were imaged using a ChemiDoc Touch MP system (Bio-Rad Laboratories, Hercules, CA, USA). Bands were quantified using ImageJ, and statistical data were analyzed using GraphPad Prism 9.
6
Molecular Profiling of Cancer Biomarkers
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PureLink RNA isolation kit, RevertAid First Strand cDNA Synthesis Kit, DNase I enzyme, PowerUp SYBR Green Master Mix were purchased from Thermo Fisher Scientific (Waltham, Massachusetts, United States). RNAlater, TRIzol Reagent and Bradford reagent were purchased from Sigma-Aldrich (St. Louis, Missouri, United States). Primers designed for the genes were purchased from Integrated DNA Technologies (Coralville, Iowa, United States). Primary antibodies against SPHK1 (NBP2-20472); CERK (NB100-2911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States). Monoclonal primary antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (AM4300) was received from Thermo Fisher Scientific (Waltham, Massachusetts, United States). HRP-linked anti rabbit secondary antibody (7074) was purchased from Cell Signaling Technology (Danvers, Massachusetts, United States) and HRP- linked anti-mouse (SC-2005) was obtained from Santa Cruz Biotechnology (Dallas, Texas, United States). Clarity ECL Western Blotting Substrate was purchased from Bio-Rad (Hercules, CA, USA). All other chemicals used were of molecular biology grade.
7
Investigating T-ALL Cell Signaling Pathways
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T-ALL cell lines were cultured in the presence or absence of 1 μM OP449, 1 μM dovitinib, or combination. Following the indicated drug exposure time, cells were washed in PBS and lysed in 50 μL lysis buffer (9803, Cell Signaling Technology, Boston, MA) supplemented with complete protease inhibitor and phosphatase inhibitor cocktail-2 (Sigma-Aldrich, St. Louis, MO). Equal amounts of protein were fractionated on 4–15% Tris-glycine polyacrylamide gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes and probed with antibodies to SETBP1 (98222) and p-cMYC (78318) from AbCam, San Francisco, CA; c-MYC (764) from Santa Cruz, Dallas, TX; p-AKT (4060), AKT (9272), p-ERK1/2 (9101), ERK1/2 (4695), p-p70S6K (9234), and p70S6K (2708) from Cell Signaling, Danvers, MA; p-Tyr (05-321), actin (C4), and PP2A-C (05-421) from Millipore, Temecula, CA; SET (A302-261a) from Bethyl, Montgomery, TX; CIP2A (a gift from Jukka Westermarck, Finnish Cancer Institute, Turku Centre for Biotechnology, Turku, Finland), p-PP2A-Y307 (1155-1) from Epitomics, Cambridge, MA; and GAPDH (AM4300) from ThermoFisher, Carlsbad, CA.
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