Gapdh sc 137179

Manufactured by Santa Cruz Biotechnology

Sourced in United States

GAPDH: sc-137179 is a primary antibody product manufactured by Santa Cruz Biotechnology. It is designed to detect the expression of the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein, which is a critical enzyme involved in the glycolytic pathway.

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GAPDH (3335 citations) Sc-137179 (10 citations) Mouse anti-human GAPDH monoclonal antibody (sc-137179 (2 citations)

5 protocols using gapdh sc 137179

Quantitative Western Blotting of SGPL1

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Western blotting procedure was performed as described previously [27 (link)]. Recombinant protein of human sphingosine-1-phosphate lyase 1 (SGPL1) was produced with TrueORF clone, RC208705 encoding the full-length human SGPL1 with C-terminal DDK tag, from human HEK293 cells (GenBank accession: NM_003901, Predicted molecular weight: 63.3 kDa, purchased from OriGene Technologies, Inc.Rockville, USA; CAT#: TP308705. For protein detection, primary antibodies (MaxPab^®; ABIN948744, USA; β-Actin #4970: Cell Signaling, USA; GAPDH: sc-137179, Santa Cruz, USA) were incubated overnight at 4°C followed by labeling with a horseradish peroxidase (HPR)-conjugated secondary antibody (Cell Signaling, USA) for 1 hour at room temperature. Protein signals were visualized by using SuperSignal West Femto Chemiluminescent Substrate (Pierce Biotechnology, Rockford, USA) for detection of peroxidase activity from HRP-conjugated antibodies. Band intensity was analyzed densitometrically with the Molecular Imager ChemiDoc XRS and Image Lab 3.0.1 software (Bio-Rad, USA). Protein detection was repeated at least three times with individually prepared cell lysates from independent passaged cells.

Engel N., Adamus A., Frank M., Kraft K., Kühn J., Müller P., Nebe B., Kasten A, & Seitz G. (2018). First evidence of SGPL1 expression in the cell membrane silencing the extracellular S1P siren in mammary epithelial cells. PLoS ONE, 13(5), e0196854.

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Protein Extraction and Immunoblotting

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Adult heads were dissected and homogenized in a protein extraction buffer. For cell lines, 10⁷ cells were lysed in RIPA buffer. The following primary antibodies were used: c-Abl (sc-23), Dab1 (sc-271136), p-Tyr (sc-7020), GAPDH (sc-137179) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-tubulin (CP06; Oncogene Research Products, Merck KGaA, Darmstadt, Germany) mouse monoclonal antibodies, BCR (sc-20707) rabbit polyclonal antibody (Santa Cruz Biotechnology) and mouse 5G2 anti-Enabled supernatant (Developmental Studies Hybridoma Bank - DSHB, University of Iowa, IA, USA). For immunoprecipitation, 1 mg of total protein extract was incubated with anti-Enabled supernatant and subsequently with protein A sepharose (Amersham Bioscience, GE Healthcare, Waukesha, WI, USA) (Online Supplementary Data).

Bernardoni R., Giordani G., Signorino E., Monticelli S., Messa F., Pradotto M., Rosso V., Bracco E., Giangrande A., Perini G., Saglio G, & Cilloni D. (2019). A new BCR-ABL1 Drosophila model as a powerful tool to elucidate the pathogenesis and progression of chronic myeloid leukemia. Haematologica, 104(4), 717-728.

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NF-κB DNA-binding Activity Assay

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Immunoblotting was performed as previously described.^{19 (link)} The following antibodies were used: p100 (sc-7386), p105 (sc-7178), Rel-B (sc-226), Rel-A (sc-372), p53 (2876) and GAPDH (sc-137179), all from Santa Cruz Biotechnology, TX. Histone H3 (9715) antibody was purchased from Cell Signaling Technology, MA. Nuclear and cytoplasmic fractions were obtained following the nuclear extraction protocol (Active Motif, CA).
DNA-binding activity of NF-κB in the OCI-LY3 cell line was assessed using a supershift assay. Double-stranded consensus oligonucleotide sequences representing the NF-κB response element (5′-AGTTGAGGGGACTTTCCCAGGC-3′ and 3′-TCAACTCCCCTGAAAGGGT CCG-5′) were purchased from Promega, WI. Primers were labeled with γ³²P-ATP using T4 kinase. After the labeling reaction, the mixtures were cleared with G-50 minicolumns. Ten micrograms of OCI-LY3 nuclear extract was incubated overnight with the radiolabeled probes and 2 μL Rel-A or Rel-B antibody and electrophoresed at 4°C (150 V) for 90 min on a 5% polyacrylamide gel containing 50 mM Tris, pH 7.5, 0.38 M glycine and 2 mM EDTA. The gels were then processed for autoradiography.

Ramachandiran S., Adon A., Guo X., Wang Y., Wang H., Chen Z., Kowalski J., Sunay U.R., Young A.N., Brown T., Mar J.C., Du Y., Fu H., Mann K.P., Natkunam Y., Boise L.H., Saavedra H.I., Lossos I.S, & Bernal-Mizrachi L. (2014). Chromosome instability in diffuse large B cell lymphomas is suppressed by activation of the noncanonical NF-κB pathway. International journal of cancer, 136(10), 2341-2351.

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Western Blot Analysis of Signaling Proteins

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Cells were harvested using radio-immunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Bimake, Houston, TX). Equal amounts of protein were loaded per lane into an SDS-PAGE followed by transferring onto a nitrocellulose membrane (BioRad). The blots were blocked with 5% nonfat dried milk followed by incubation in the respective antibodies. After several washes, membranes were incubated with appropriate secondary antibodies and imaged using either chemiluminescence or the LICOR Odyssey Infrared Imaging System (Lincoln, NE). Antibodies for western blot analysis were used at 1:1000 dilution unless specified otherwise. HA-Tag #sc-7392, Fra-1 #sc-183, ATF-2 #sc-187, GAPDH #sc-137179 were from Santa Cruz Biotechnology; anti-phosphor-MK2 #07-155 from Upstate Biotechnology; Myc-tag #2866, ERK #9102, phosphor-ERK #4376, p38^MAPK #9212, phosphor-p38^MAPK #4511, JUN #9165, MK2 #3042, Jab1 #9444, Cyclin D1 #2922, βActin #4967 were from Cell Signaling; FLAG #F3165 was from MilliporeSigma; uPA #395 and uPAR #3937 were from American Diagnostica used at 1:500 dilution; Anti-phosphorylated JAB1 (Ser¹⁷⁷) pAb was commercially prepared by EZ Biolab (Carmel, IN) using synthesized AVVIDPTRTI(pS)AGKVN peptide and used at 1:500 dilution. All blots derive from the same experiment and were processed in parallel.

Chen H., Padia R., Li T., Li Y., Li B., Jin L, & Huang S. (2021). Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1. NPJ Breast Cancer, 7, 91.

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Protein Analysis of Lung and Fibroblast Samples

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Protein was extracted from lung tissues and fibroblasts with RIPA buffer (containing 0.1% PMSF), and equal amounts of protein from each sample (50 µg)
were separated by 10% SDS/PAGE and transferred to polyvinylidene fluoride membranes. The membranes were then incubated with primary antibodies overnight at 4°C, and horseradish peroxidase (HRP)-coupled goat anti-mouse or goat anti-rabbit secondary antibody (sc-2005, 1:2,000, sc-2030, 1:5,000; Santa Cruz, CA, USA). The chemiluminescence signals were detected with the EasySee Western Blot Kit (Beijing TransGen Biotech, Beijing, China).The densitometric analysis was conducted with Image J 1.43 (National Institutes of Health). Primary antibodies against eIF3a (#3411, 1:1,000), phospho-ERK1/2 (#9106, 1:2,000), ERK1/2(#9107, 1:1,000) were D r a f t purchased from Cell Signaling (Boston, MA, USA). Primary antibodies against α-SMA (ab5694, 1:2000), TGF-β 1 (ab27969, 1:500), collagen I (ab34710, 1:1000), and collagen III (ab7778, 1:1000) was purchased from Abcam (Hong Kong, China), and GAPDH (sc-137179, 1:2000) were obtained from Santa Cruz (CA, USA).

Li X.W., Li X.H., Du J., Li D., Li Y.J, & Hu C.P. (2016). Calcitonin gene-related peptide down-regulates bleomycin-induced pulmonary fibrosis. Canadian journal of physiology and pharmacology, 94(12).

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