Gapdh

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GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is an enzyme involved in the glycolytic pathway, a fundamental metabolic process that converts glucose into energy. GAPDH catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, playing a key role in energy production within cells.

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Lab products found in correlation

1 486 protocols using gapdh

Glucose Transporter Gene Expression Analysis

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Gene primers SGLT1, GLUT2, and GAPDH were purchased from ThermoFisher Scientific (Waltham, MA).

Gene primer	Gene Symbol	Entrez Gene ID	Gene Name	Gene Aliases	Chromosome Location	UniGene
SGLT1 (Hs01573790_m1)	SLC5A1	6523	solute carrier family 5 member 1	D22S675; NAGT; SGLT1	Chr.22: 32043032 - 32113029 on Build GRCh38	Hs.1964
GLUT2 (H01096908_m1)	SLC2A2	6514	solute carrier family 2 member 2	GLUT2	Chr.3: 170996341 - 171026979 on Build GRCh38	Hs.167584
GAPDH (Hs02758991_g1)	GAPDH	2597	glyceraldehyde-3-phosphate dehydrogenase	G3PD; GAPD; HEL-S-162eP	Chr.12: 6534405 - 6538375 on Build GRCh38	Hs.544577

2-NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) was purchased from ThermoFisher (Carlsbad, CA).

Mojica L., Luna-Vital D.A, & Gonzalez de Mejia E. (2018). Black bean peptides inhibit glucose uptake in Caco-2 adenocarcinoma cells by blocking the expression and translocation pathway of glucose transporters. Toxicology Reports, 5, 552-560.

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Quantitative RT-PCR Analysis of 5-HT3A Receptor

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Quantitative RT‐PCR was performed by TaqMan gene expression assay using ABI Prism 7900 sequence detection system with a 384‐well format, according to the manufacturer's protocol. 5‐HT3A receptor mRNA level was determined as previous described (Li et al., 2014). In brief, human Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH, Applied Biosystems 1997, CA, USA) was used as an endogenous control to normalize the mRNA expression level of target genes. All specific primers, TaqMan probes/primers for target gene and GAPDH were obtained from Applied Biosystems. Quantitative RT‐PCR reaction was as follows: 50° C for 2 min and 95° C for 10 min, then 95° C for 50 cycles of 10s, 59° C for 1 min. Each subject was repeated in triplicate. The experiment performers were blind to all the clinic data.

Chen H., Fan Y., Zhao L., Hao Y., Zhou X., Guan Y, & Li Z. (2017). Successful treatment with risperidone increases 5‐HT 3A receptor gene expression in patients with paranoid schizophrenia – data from a prospective study. Brain and Behavior, 7(9), e00798.

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Quantitative Real-Time PCR Analysis of Stem Cell Markers

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Preparation of cDNA from mRNA was performed directly from cultured cell lysate using the TaqMan^® Gene Expression Cells-to-CT™ kit (Ambion, Japan), according to the manufacturer's instructions. Cell lysate were reverse transcribed to cDNA using the Reverse Transcription (RT) Enzyme Mix and appropriate RT buffer (Ambion). Finally the cDNA was amplified by quantitative real-time PCR (qRT-PCR) using the included TaqMan Gene Expression Master Mix and the specific TaqMan primer/probe assay designed for the investigated genes: ALDH1A1 (Hs00946916_m1), NANOG (Hs02387400_g1), OCT4 (Hs03005111_g1), SOX2 (Hs01053049_s1) and GAPDH (Hs99999905_m1), (Applied Biosystems, Tokyo, Japan).
The gene expression levels were normalized to the expression of the housekeeping gene GAPDH and were expressed as fold changes relative to the expression of the untreated cells. The amplification profile was initiated by 10-min incubation at 95°C, followed by two-step amplification of 15 sec at 95°C and 60 sec at 60°C for 40 cycles. All experiments were performed including non-template controls to exclude contamination of the reagents. Quantification was done with the delta/delta Ct calculation method (17 (link)). Each sample was analyzed in triplicate on the 7500 Real-Time PCR system in a 96-well plate (Applied Biosystems).

YATA K., BEDER L.B., TAMAGAWA S., HOTOMI M., HIROHASHI Y., GRENMAN R, & YAMANAKA N. (2015). MicroRNA expression profiles of cancer stem cells in head and neck squamous cell carcinoma. International Journal of Oncology, 47(4), 1249-1256.

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Tocilizumab Modulates IL-6 Response in Monocytes

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Human monocytes from healthy donors were pre-incubated with or without 0.5 mg/mL tocilizumab and then incubated with or without recombinant human IL-6 (0.1ug/ml), normal donor plasma, or patient plasma for an additional 30 minutes in IMDM supplemented with 1.0% human A/B serum. Tocilizumab (Actemra™, Genentech) was purchased through the Hospital of the University of Pennsylvania Pharmacy. Following stimulation, culture medium was removed, cells were lysed directly in TRIzol, and RNA was isolated using a Qiagen RNeasy Kit per manufacturer instructions. cDNA was synthesized from 0.5 – 1.0 μg of RNA per sample (Applied Biosystems, Foster City, CA), and primers for qRT-PCR were designed using the Primer 3 online program (25 (link), 26 (link)) and synthesized by Integrated DNA Technologies (SOCS3) or purchased from Applied Biosystems (GAPDH). Relative quantification was measured using either SYBR Green chemistry (Applied Biosystems; SOCS3) or Taqman chemistry (Applied Biosystems; GAPDH). SOCS3 expression was normalized to GAPDH and relative expression was calculated using the ΔCT formula. The fold increase or decrease in expression of treated samples relative to no treatment controls was calculated (ΔΔCT). Primer sequences for human SOCS3 are as follows: (i) Forward: 5′-CAAGGACGGAGACTTCGATT-3′, (ii) Reverse: 5′-AACTTGCTGTGGGTGACCAT-3′.

Long K.B., Tooker G., Tooker E., Luque S.L., Lee J.W., Pan X, & Beatty G.L. (2017). IL-6 receptor blockade enhances chemotherapy efficacy in pancreatic ductal adenocarcinoma. Molecular cancer therapeutics, 16(9), 1898-1908.

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Quantifying Gene Expression in Tissues

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RNA from tissues and cells was extracted using TRIzol (Invitrogen) and the RNeasy Mini Kit (Qiagen). RNA integrity was checked by 2100 Bioanalyzer (Agilent Technologies). qRT‐PCR was performed using High capacity cDNA Reverse Transcription Kit, TaqMan Universal PCR Master Mix, and TaqMan gene expression assays (Rattus norvegicus: MMP‐2 (Rn01538170_m1), MMP‐3 (Rn00591740_m1), MMP‐9 (Rn00579162_m1), MMP‐13 (Rn01448194_m1), GAPDH (Rn99999916_s1); Mus musculus: MMP‐9 (Mm00442991_m1), GAPDH (Mm99999915_g1), NKp46 (Mm01337324_g1), F4/80 (Mm00802529_m1), CD3z (Mm00446171_m1), CD4 (Mm00442754_m1), CD8a (Mm01182107_g1), HPRT1 (Mm01545399_m1) from Applied Biosystems.

Jenke A., Schur R., Röger C., Karadeniz Z., Grüger M., Holzhauser L., Savvatis K., Poller W., Schultheiss H., Landmesser U, & Skurk C. (2017). Adiponectin attenuates profibrotic extracellular matrix remodeling following cardiac injury by up‐regulating matrix metalloproteinase 9 expression in mice. Physiological Reports, 5(24), e13523.

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Quantitative Analysis of Hepatic IGF1 and IGFBP3

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After sacrifice, the liver was immediately removed, rinsed, and stored at −80°C. Total RNA was extracted from hepatocytes by QIAzol (Qiagen, USA) and transcribed into cDNA using the transcription kit (AppliedBiosystems, USA). Quantitative PCR was performed with a real-time PCR system (AppliedBiosystems, USA) followed by 40 cycles of 95 °C for 10 min, 95 °C for 15 s, and 60 °C for 60 s. Primer was designed in Bioneer (Korea): IGF1 (GenBank M15481), forward 5′-GCTATGGCTCCAGCATTCG-3′ and reverse 5′-TCCGGAAGCAACACTCATCC-3′; IGFBP3 (GenBank NM_012588), forward 5′-GGAAAGACGACGTGCATTG-3′ and reverse 5′-GCGTATTTGAGCTCCACGTT-3′; and glyceraldehyde−3−phosphate dehydrogenase (GAPDH, GenBank NM_017008), forward 5′-TGGCCTCCAAGGAGTAAGAAAC-3′ and reverse 5′-CAGCAACTGAGGGCCTCTCT-3′. Conditions were repeated 40 times at 95 °C for 10 m, 95 °C for 15 s, 60 °C for 60 s. Relative quantification was performed by normalizing using GAPDH expression using the delta-delta Ct method.

Lee D., Lee S.H., Cho N., Kim Y.S., Song J, & Kim H. (2019). Effects of Eleutherococcus Extract Mixture on Endochondral Bone Formation in Rats. International Journal of Molecular Sciences, 20(5), 1253.

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Quantitative Real-Time RT-PCR Analysis

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The real-time RT-PCR method with an Assay-on-Demand Gene Expression Product (Life Technologies, P/N 4331182) consisted of unlabeled PCR primers and a TaqMan MGB probe (FAM dye-labeled) optimized to work with the TaqMan Universal PCR Master Mix (P/N 4304437) in an ABI Prism 7700 system (PerkinElmer Life Sciences, Boston, MA, USA) and was employed to quantitatively measure transforming growth factor alpha (TGFA; Hs00608187_m1), transforming growth factor beta 1 (TGFB1; Hs00998133_m1), progesterone receptor (PGR; Hs01556702_m1), tumor necrosis factor receptor superfamily member 9 (TNFRSF9; Hs00155512_m1), bone morphogenetic protein 6 (BMP6; Hs01099594_m1), thyroid hormone receptors α/β (THRA; Hs00268470_m1, and THRB; Hs00230861_m1), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Hs02758991_g1) mRNA expression (Applied Biosystems). All assays were performed in triplicate. The mRNA contents were normalized to GAPDH mRNA levels, and differences in expression were determined by the CT method described in the ABI user's manual (Life Technologies).

Conde S.J., Luvizotto R.D., de Síbio M.T, & Nogueira C.R. (2014). Thyroid Hormone Status Interferes with Estrogen Target Gene Expression in Breast Cancer Samples in Menopausal Women. ISRN Endocrinology, 2014, 317398.

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Quantitative Gene Expression Analysis in Neurons

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Total RNA from primary neurons was isolated as previously described (75 (link)). Mouse hippocampi were homogenized in the RLT buffer from RNeasy Mini Kit (Qiagen, 74106) with a tissue grinder gun and further processed according to the manufacture protocol for RNA extraction. Retro transcription was done using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). mRNA copy numbers relative to Gapdh mRNA levels were measured by real-time PCR using Taqman Universal MasterMix (Applied Biosystems, USA) and the following primers: Esr1, Esr2, Arc, Bdnf, Cyp19a1, Sdr5a1, Hsd17b10, Hsd17b1, Rara, Cyp26b1, Srepb1, Rxrg, and Gapdh (Applied Biosystems, USA).

Latorre-Leal M., Rodriguez-Rodriguez P., Franchini L., Nikolidakis O., Daniilidou M., Delac L., Varshney M.K., Arroyo-García L.E., Eroli F., Winblad B., Blennow K., Zetterberg H., Kivipelto M., Pacciarini M., Wang Y., Griffiths W.J., Björkhem I., Matton A., Nalvarte I., Merino-Serrais P., Cedazo-Minguez A, & Maioli S. (2024). CYP46A1-mediated cholesterol turnover induces sex-specific changes in cognition and counteracts memory loss in ovariectomized mice. Science Advances, 10(4), eadj1354.

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Gene Expression Analysis of Human Liver Cells

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Total RNA was isolated from human liver epithelial THLE-3 cells by using TriPure® Isolation Reagent (Roche, Indianapolis, USA). The integrity of the extracted RNA was confirmed with Bioanalizer (Agilent, Palo Alto, CA, USA). The reverse transcription was performed in 300 ng of total RNA with TaqMan reverse transcription reagents kit (Applied Biosystems, Perkin-Elmer Corporation, CA, USA). cDNA was amplified with specific primers and probes predesigned by Applied Biosystems for α-SMA (Hs00559403_m1), α₁(I)-collagen (Col type I; cat. n°: Hs00164004_m1), vimentin (cat. n°: Hs 00958116_m1), E-cadherin (cat. n°: Hs01023894_m1), zona occludens-1 (ZO-1; cat. n°: Hs01551861_m1), Slug (cat. n°: Hs00950344_m1), Snail (cat. n°: Hs00195591_m1) and GAPDH (pre-designed by Applied Biosystems, cat. n°: 4310884E) as a housekeeping in a 7900HT Fast Real-Time PCR System (Applied Biosystem) using Universal Master Mix (Applied Biosystems). Relative quantification of these different transcripts was determined with the 2^−ΔΔCt method using GAPDH as endogenous control (Applied Biosystems; 4352339E) and normalized to control group.

Pastor-Clerigues A., Marti-Bonmati E., Milara J., Almudever P, & Cortijo J. (2014). Anti-Inflammatory and Anti-Fibrotic Profile of Fish Oil Emulsions Used in Parenteral Nutrition-Associated Liver Disease. PLoS ONE, 9(12), e115404.

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Quantifying c-MET Expression in Tissues

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Total RNA, including mRNA, was extracted from the tissues using the mirVana miRNA isolation kit (Ambion, Texas, USA). cDNA was synthesized from total RNA using oligo dT primer (Invitrogen, San Diego, USA) in accordance with the manufacturer’s TaqMan RNA assay protocol (PE Applied Biosystems, San Francisco, USA). Real-time PCR was performed using an Applied Biosystems 7900HT Sequence Detection system (Applied Biosystems) in accordance with the manufacturer’s protocol. Data normalization was performed using GAPDH. To avoid amplification of genomic DNA, the primers and probes for amplifying c-MET (Met) and GAPDH were chosen to hybridize at the junction between the 2 exons as follows: c-MET (Met) (Hs1565584_m1; Applied Biosystems) and GAPDH (#4310884E; Applied Biosystems). Relative quantification of c-MET expression was calculated using the 2^−ΔΔC_T method.

Kim H.J., Yoon A., Ryu J.Y., Cho Y.J., Choi J.J., Song S.Y., Bang H., Lee J.S., Cho W.C., Choi C.H., Lee J.W., Kim B.G, & Bae D.S. (2016). c-MET as a Potential Therapeutic Target in Ovarian Clear Cell Carcinoma. Scientific Reports, 6, 38502.

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