Total tissue lysate (TTL) from harvested mouse orthotopic tongue tumors was prepared by grinding tumors to a fine powder in liquid nitrogen and extracting total tissue proteins with Triton/β-octylglucoside buffer. Protein concentrations for TTL were determined using BCA assay (Pierce). For immunoblot analyses, TTL (20–30 μg of total protein) were fractionated on 7.5% SDS-PAGE, transferred onto polyvinylidene difluoride membranes, blocked with 5% nonfat dry milk, and incubated with primary antibodies to either E-cadherin or β-catenin (Abcam, rabbit) and GAPDH (Novus Biologicals). Protein-specific detection was carried out with horseradish peroxidase-labeled secondary antibodies conjugated to horseradish peroxidase (Bio-Rad) and Enhanced Chemiluminescence Plus (Amersham Biosciences). Signal intensities were normalized to GAPDH (Novus Biologicals).
Gapdh
Manufactured by Novus Biologicals
Sourced in United States
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is an enzyme involved in the glycolytic pathway, which is responsible for the conversion of glucose to energy. GAPDH catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
β actin, by Merck Group (5 mentions)
P akt, by Cell Signaling Technology (4 mentions)
Caspase 3, by Novus Biologicals (4 mentions)
β actin, by Novus Biologicals (3 mentions)
Caspase 8, by Cell Signaling Technology (3 mentions)
Caspase 9, by Cell Signaling Technology (3 mentions)
Ripa buffer, by Cell Signaling Technology (3 mentions)
Vinculin, by Merck Group (3 mentions)
Arl13b, by Proteintech (3 mentions)
Ccl26, by R&D Systems (2 mentions)
Laminin, by Merck Group (2 mentions)
Nb300 221, by Novus Biologicals (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Anti phospho histone h2a x ser139, by Merck Group (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Hsp60, by Santa Cruz Biotechnology (2 mentions)
Ndp52, by BD (2 mentions)
84 protocols using gapdh
1
Immunoblot Analysis of Mouse Tongue Tumor Proteins
2
Western Blotting of BIRC3, NF-κB Pathway
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Cells were harvested in the RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) and the protein concentration of each sample was determined using BCA protein assay reagent kit (Thermofisher Scientific, Inc) after transfection of 48 h. The supernatants containing total protein were mixed with corresponding volume of 5×SDS loading buffer (Beyotime Biotechnology, Shanghai, China) and heated at 100 °C for 10 min. The lysates of 20 μg were electrophoresed on 10% SDS-PAGE and transferred to PVDF membranes. The proteins were blocked containing 5% defatted milk for 1 h at room temperature. Subsequently, primary antibodies were incubated at 4 °C overnight, which consisted of the following: BIRC3 (Abcam, cat: ab32059, 1:1000); p-P65 (Abcam, cat: ab6503, 1:1000); P65 (Abcam, cat: ab16502, 1:1000); IκBα (CST, cat: #4812, 1:1000); p-IκBα (CST, cat: #2859, 1:1000); c-myc (CST, cat: 5605, 1:1000); GAPDH (Novus Biologicals, cat: 2D4A7, 1:5,000). Followed by incubation with the horseradish peroxidase conjugated secondary antibodies (anti-rabbit, 1:10,000; cat: #7074; or anti-mouse, 1:10,000; cat: #7076; both from CST, Inc.) for 1.5 h at room temperature. To ensure that equal amounts of sample protein were applied for electrophoresis, GAPDH was used as an internal control.
3
Comprehensive Protein Analysis Protocol
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Cells were lysed in SDS lysis buffer (240 mM Tris-acetate, 1% SDS, 1% glycerol, 5 mM EDTA pH 8.0) with DTT, protease inhibitors, and a cocktail of phosphatase inhibitors. Antibody against vimentin, and MCP-1 were purchased from Abcam (Cambridge, MA, USA). Antibodies detecting CD44 and phospho-YAP S127 were purchased from GeneTex, Inc. (Irvine, CA, USA). Antibodies against YAP, phospho-MST1/MST2 T183/T180, MST1, phospho-LATS1 S909, LATS1, CYR61, CTGF, phospho–AKT T308, AKT, and phospho–GSK3β S9, phospho-ERK1/2, ERK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). The β-actin, GAPDH, and α -tubulin antibodies were purchased from Novus Biologicals (Littleton, CO, USA). Antibody against E-cadherin was purchased from BD (Franklin Lakes, NJ, USA).
4
Immunoblotting of Glutamate Receptors
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Protein lysates were collected by scraping cells in RIPA lysis buffer (Santa Cruz, CA). Protein (30 to 100 µg) was separated by SDS-polyacrylamide gel electrophoresis (4 to 20%) and transferred to polyvinylidene fluoride membranes. Immunodetection of mGluR1 (BD Pharmagen, San Jose, CA), and phosphorylated or total ERK (Cell Signaling, Canton, MA), was performed using primary antibodies to these antigens with appropriate secondary antibodies followed by detection using chemiluminescence. Primary blots were stripped and reprobed with antibody against α-tubulin (Sigma-Aldrich, St. Louis, MO) or GAPDH (Novus Biologicals, Littleton, CO).
5
Annexin V-AF568 and Hoechst 33342 Staining
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Annexin V-AF568 and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). O-phospho-L-serine (OPS) and Phorbol 12-myristate-13-acetate (PMA) were obtained from Sigma Aldrich (St. Louis, MO, USA). Ionomycin (IO) was purchased from Merck Millipore (Darmstadt, Germany). Hydroxamate-based ADAM17/ADAM10 inhibitor GW280264X [30 (link)] was purchased from Aeobious (Gloucester, MA, USA). Marimastat and ADAM10 inhibitor GI254023X [31 (link)] were purchased from Tocris Bioscience (Bristol, UK). Additional antibodies used: ANO1 (Bio-Techne, Abingdon, UK), ANO4 (Aviva Systems Biology, San Diego, CA, USA), ANO5 (Abcam, Cambridge, UK), ANO6 and ANO7 (OriGene, Rockville, MD, USA), ANO9 and ANO10 (LSBio, Seattle, WA, USA), and GAPDH (Novus Biologicals, Littleton, CO, USA). Secondary antibodies for automated Western were obtained from Protein Simple (San Jose, CA, USA) or Novus Biologicals (Novus Biologicals, Littleton, CO, USA).
6
Quantitative Western Blot Analysis
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After running the NuPAGE, proteins were transferred to PVDF membranes (BioRad Laboratories Inc, Hercules, CA) which were blocked in SuperBlock (T20 TBS buffer, Thermo Fisher) for one hour. For secreted proteins, equal loading was confirmed by Ponceau staining of the membranes79 (link). The PVDF membranes were incubated in a polyclonal rabbit anti-azurin antibody1 (link) (1:5000) or a monoclonal mouse anti-aldolase A (1:200, Santa Cruz Biotechnology, TX) at 4 °C overnight. For cell lysates, GAPDH was used as a loading control (Novus Biologicals). The secondary antibody was applied (1:1000, polyclonal goat anti-rabbit IgG-HRP; Santa Cruz Biotechnology). The signal was detected using enhanced chemiluminescence (ECL, Thermo Fisher Scientific). Quantitative analysis of the bands was performed with a computer-assisted imaging densitometer (UN-SCAN-IT gel version 5.1).
7
Western Blot Analysis of Apoptosis Regulators
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Specific protein expression in cell lines was determined by Western blot analysis as described before [18 (link)] using the following primary antibodies: Mcl-1 (1:500; CST: Cell Signaling Technology, Danvers, MA), human caspase 9 (1:1,000; CST), cleaved caspase 3 (1:250; CST), cleaved PARP (Asp214, 1:1000; CST), Bcl-xL (1:500; CST), Usp9X (1:1000; CST), XIAP (1:1000; CST), Survivin (1:1000; CST), BIM (1:500; CST), Noxa (1:500, clone 114C307; Calbiochem), β-actin (1:2,000, clone AC15; Sigma Aldrich), Bag3 (1:500; Abcam, Cambridge, MA). 14-3-3 (1:1,000, SCB: Santa Cruz Biotechnology), GAPDH (1:1000, clone 1D4, Novus Biologicals) and secondary HRP-linked antibodies were purchased from SCB.
8
SDS-PAGE and Western Blot Analysis
9
Combination Treatment Induces Apoptosis in Pancreatic Cancer
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MIA PaCa-2 and PANC-1 cells were seeded and treated with 0.4 µM CM03, SAHA (1 µM for MIA PaCa-2 and 4 µM for PANC-1) or in combination of both compounds, at these concentrations. The treatment continued for 24 h in MIA PaCa-2 cells and for 48 h in PANC-1 cells because 24 h treatment was insufficient for observing its effect on PANC-1 cells. Untreated or vehicle treated (DMSO) cells were used as a control. Cells were collected and lysed with RIPA lysis buffer (ThermoFisher, Cat. No. 89900) supplemented with protease and phosphatase inhibitors (ThermoFisher, Cat. No. 78442). The lysate concentration was quantified using the Pierce™ BCA Protein Assay Kit (ThermoFisher, Cat. No. 23227). Capillary-based automated Western blotting was performed on a Wes machine (https://www.proteinsimple.com/wes.html ) to run the cell lysates and detect proteins of interest according to the manufacturer’s instructions. Antibodies used were for cleaved PARP (Cell Signaling, Cat. No. 5625), γ-H2AX (Novus Biologicals, Cat. No. NB100-384), and GAPDH (Novus Biologicals, Cat. No. NB300-325).
10
Western Blot Analysis of eNOS and p-eNOS
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Total eNOS and p-eNOS protein levels were determined in LV homogenates as previously described [43 (link)], and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Novus Biologicals, Littleton, CO, USA)).
Subsequent to SDS-PAGE, the proteins were transferred to nitrocellulose membranes, and the blots were probed with primary anti-eNOS (1:500, BD Transduction Laboratory, San Jose, CA, USA), anti-p-eNOS (1:1000 Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:10,000, Imgenex), and secondary rabbit anti-mouse immunoglobulin G (IgG) antibody conjugated with horseradish peroxidase (HRP; 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). All blots were analyzed using the Odyssey system (LI-COR, Lincoln, NE 68504, USA).
Subsequent to SDS-PAGE, the proteins were transferred to nitrocellulose membranes, and the blots were probed with primary anti-eNOS (1:500, BD Transduction Laboratory, San Jose, CA, USA), anti-p-eNOS (1:1000 Cell Signaling, Danvers, MA, USA), anti-GAPDH (1:10,000, Imgenex), and secondary rabbit anti-mouse immunoglobulin G (IgG) antibody conjugated with horseradish peroxidase (HRP; 1:1000, Santa Cruz Biotechnology, Dallas, TX, USA). All blots were analyzed using the Odyssey system (LI-COR, Lincoln, NE 68504, USA).
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