Mouse anti gapdh

Manufactured by Biosynth

Sourced in United States

Mouse anti-GAPDH is a primary antibody that specifically binds to the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein. GAPDH is a commonly used housekeeping gene and is involved in the glycolytic pathway. This antibody can be used to detect and quantify GAPDH expression in various cell and tissue samples.

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Lab products found in correlation

Mouse anti p53, by Santa Cruz Biotechnology (2 mentions) Adi spa 810 d, by Enzo Life Sciences (1 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions) Tris glycine polyacrylamide gel, by Thermo Fisher Scientific (1 mentions) Protease inhibitor, by Roche (1 mentions) Horse serum, by Merck Group (1 mentions) Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions) Non targeting control sirna, by Horizon Discovery (1 mentions) Mouse anti myc antibody, by Santa Cruz Biotechnology (1 mentions) Rabbit anti orai1 antibody, by Alomone (1 mentions) Rabbit anti calreticulin, by Abcam (1 mentions) Fura 2 am, by Thermo Fisher Scientific (1 mentions) L glutamine, by Merck Group (1 mentions) Quikchange xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Immobilon p polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions) Peroxidase affinipure goat anti mouse, by Jackson ImmunoResearch (1 mentions) Peroxidase affinipure goat anti rabbit, by Jackson ImmunoResearch (1 mentions) Pierce bicinchoninic acid bca assay kit, by Thermo Fisher Scientific (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Anti trx1, by Cell Signaling Technology (1 mentions)

10 protocols using mouse anti gapdh

Quantifying HSP70 and GAPDH Protein Levels in A549 Cells

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A549 cells were seeded in six-well plates at 4.0 × 10⁵ cells/well and allowed to attach overnight. The next day, medium was aspirated and replaced with 1.0 mL fresh growth medium and treated with indicated drug. After 6 hours, medium was aspirated and the cells were harvested, quantified, and transferred to nitrocellulose membranes as described[20 (link)]. Primary antibodies were used at a dilution of 1:1500 mouse anti-HSP70 (Enzo,ADI-SPA-810-D, clone: C92F-3A-5) and 1:10000 mouse anti-GAPDH (Fitzgerald Industries®, cat# 10R-G109a). Species-specific europium-conjugated secondary antibody (ScanLater goat anti mouse; part#: R7562; Molecular Devices®) was used. Afterward, membranes were washed with TBS-T and analyzed via Spectramax i3x® western blot cartridge (Molecular Devices®). Two independent experiments were performed in triplicate (n=6).

Bacon N.A., Larre I., Lawag A.A., Merritt C I.I., Smith M., Rosolen M, & Sollars V.E. (2020). Low Dose HSP90 Inhibition with AUY922 Blunts Rapid Evolution of Metastatic and Drug Resistant Phenotypes Induced by TGF-β and Paclitaxel in A549 cells. Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie, 129, 110434.

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Western Blot Analysis of Plectin

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Cells were lysed using RIPA lysis buffer that was supplemented with 1× protease inhibitor (Roche) and the lysates were collected using cell scrapers. The lysates were mixed with 2× reducing sample buffer (made with 0.125M Tris, 4% SDS, 10% 2-ME, 20% glycerol, and 0.05% bromophenol blue) and heated to 95°C for 5 min on a heating block. These samples were then loaded onto a 4–12% Tris-glycine polyacrylamide gel (Thermo Fisher Scientific) and then transferred to a nitrocellulose membrane (Thermo Fisher Scientific). The primary antibodies used for Western blotting were rabbit anti-plectin (Abcam) and mouse anti-GAPDH (Fitzgerald). The secondary antibodies used were goat anti-rabbit IgG Alexa 680 or 800 and goat anti-mouse 680 or 800 (Thermo Fisher Scientific). All Western blots were imaged using a LI-COR scanner. All blots were analyzed by comparing the intensity of the control GAPDH to the intensity of plectin.

Marks P.C., Hewitt B.R., Baird M.A., Wiche G, & Petrie R.J. (2022). Plectin linkages are mechanosensitive and required for the nuclear piston mechanism of three-dimensional cell migration. Molecular Biology of the Cell, 33(12), ar104.

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Plasmid Constructs and Reagents for SOCE Studies

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The pGW1-Myc-ATL1-wt and pGW1-Myc-ATL1-K80A plasmids15 (link) and GFP-Orai1-E106A and GFP-Orai1–R91W were described previously48 (link). The SPG3A mutants, including Myc-ATL1-Y196C, R217Q, and P342S, were created by exchanging the corresponding codons using the QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies).
TG, 2-APB, and BTP2 were purchased from VWR. NGF and Fura-2 AM were purchased from Life Technologies. Horse serum and L-glutamine were purchased from Sigma. Fetal bovine serum was purchased from Omega. Rabbit anti-calreticulin, anti-ATL2, and anti-ATL3 antibodies were purchased from Abcam. Mouse anti-Myc antibody was purchased from Santa Cruz Biotechnology. Rabbit anti-Orai1 antibody was purchased from Alomone Labs. Mouse anti-Orai1 was purchased from Abcam. Mouse anti-GAPDH was purchased from Fitzgerald. Rabbit anti-STIM1 antibody was purchased from Cell Signaling. Control non-targeting siRNA, rat STIM1 siRNA, and rat Orai1 siRNA, which includes four different siRNA oligonucleotides, were purchased from Dharmacon. ATL2 and ATL3 siRNAs used in COS-7 cells were synthesized as described previously15 (link). The cdDMEM was bought from GE. Ca0 and Ca2 solutions were prepared as described previously48 (link).

Li J., Yan B., Si H., Peng X., Zhang S.L, & Hu J. (2017). Atlastin regulates store-operated calcium entry for nerve growth factor-induced neurite outgrowth. Scientific Reports, 7, 43490.

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Protein Extraction and Western Blot Analysis

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Cells were lysed with NP-40 Lysis Buffer (1%NP-40; 200mM NaCl; 50mM Tris Base pH8.0; 10% Glycerol; in dH₂O)
or RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS) with freshly added Complete
Protease Inhibitor Cocktail (Roche). Protein concentration was determined using the Pierce bicinchoninic acid (BCA) assay kit
(ThermoFisher). Protein extracts were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and
were transferred to Immobilon-P Polyvinylidene Difluoride (PVDF) membranes (Millipore). Membranes were blocked in 5% nonfat
dry milk prepared in Tris buffered saline with 0.1% Tween-20 (TBST). Washes were performed in TBST and the following primary
and secondary antibodies diluted in 5% milk in TBST were used: mouse anti-Mdm2 (Abcam ab16895, 1:500), rabbit anti-p53 (CM5,
Leica, 1:1000), mouse anti-wild-type p53 (pAB242, provided by D. Lane and B. Vojtesek (Yewdell
et al., 1986), 1:150), mouse anti-p53 (D01, Santa Cruz sc-126, 1:1000), mouse anti-GAPDH (Fitzgerald 10R-G109a,
1:10000), Peroxidase AffiniPure goat anti-mouse (Jackson ImmunoResearch 115-035-146, 1:5000), and Peroxidase AffiniPure goat
anti-rabbit (Jackson ImmunoResearch 111-035-144, 1:5000). Immunodetection was performed using ECL Prime (Pierce).

Bowen M.E., McClendon J., Long H.K., Sorayya A., Van Nostrand J.L., Wysocka J, & Attardi L.D. (2019). The spatiotemporal pattern and intensity of p53 activation during embryogenesis dictates phenotypic diversity in p53-driven developmental syndromes. Developmental cell, 50(2), 212-228.e6.

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Antioxidant Enzyme Expression Assay

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All chemicals were purchased from Sigma (St Louis, MO) unless stated otherwise. Antibodies were purchased from following companies: mouse anti-GAPDH from Fitzgerald (Fitzgerald Industries Inc., MA, USA), rabbit polyclonal anti-Trx-1 from Cell signaling (Cell Signaling Technology Inc., MA, USA) and anti-Catalase from Sigma (Sigma Aldrich, USA). NOC18 was bought from Alexis Biochemicals (San Diego, USA). HA-tagged wild type and C152S-GAPDH genes in pRK5 expression vector were gifts from Solomon H. Snyder (John Hopkins University School of Medicine, MD).

Chakravarti R., Gupta K., Majors A., Ruple L., Aronica M, & Stuehr D.J. (2015). Novel Insights in Mammalian Catalase Heme Maturation: Effect of NO and Thioredoxin-1. Free radical biology & medicine, 82, 105-113.

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Endogenous Gene Expression Analysis

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To monitor endogenous gene responses, mouse and human organoids were harvested and centrifuged pellets were homogenized by sonication in 2× Laemmli buffer. Protein concentration of the lysates was determined using the Bradford quantification method. Homogenates were then boiled at 99°C for 5 min, and equal protein amounts were separated with 10 or 12% polyacrylamide gels and transferred to PVDF membranes using Trans-Blot Turbo System (Bio-Rad). The membranes were blocked in 5% BSA in PBS-Tween 20 and incubated for 1 hour at RT. Incubation with primary antibodies was done ON at 4°C followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Membranes were developed using ECL reagent (Thermo Fisher Scientific), and the signal was detected using ChemiDoc imager (Bio-Rad). Densiometric analysis of Western blots at nonsaturated exposure was performed using ImageJ software, and the values were normalized against the one of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. For immunoblots, the following primary antibodies at the indicated dilutions were used: rabbit anti-YAP (1:1000; Cell Signaling Technology), rabbit anti-LATS1 (1:1000; Cell Signaling Technology), and mouse anti-GAPDH (1:10000; Fitzgerald).

Rocchi C., Cinat D., Serrano Martinez P., Bruin A.L.J., Baanstra M., Brouwer U., Del Angel Zuivre C., Schepers H., van Os R., Barazzuol L, & Coppes R.P. (2021). The Hippo signaling pathway effector YAP promotes salivary gland regeneration after injury. Science signaling, 14(712).

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Synaptic Protein Regulation Analysis

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The following antibodies were used in this study: rabbit anti-phospho-cofilin (Ser³; 1:1000; Cell Signaling), rabbit anti-cofilin (1:1000; Cell Signaling Technology), rabbit anti-phospho-LIMK1 (Thr⁵⁰⁸; 1:1000; Abcam), rabbit anti-LIMK1 (1:1000; Cell Signaling Technology), rabbit anti-phospho-Slingshot1 (Ser⁹⁷⁸; 1:1000; ECM Biosciences), rabbit anti-Slingshot1 (1:1000; Abcam), rabbit anti–phospho-PAK1 (Ser¹⁹⁹; 1:1000; Abcam), rabbit anti–phospho-PAK1 (Thr⁴²³; 1:1000; Cell Signaling Technology), rabbit anti-PAK1 (1:1000; Cell Signaling Technology), mouse anti-phospho-PAK4 (Ser⁴⁷⁴; 1:1000; Santa Cruz Biotechnology), rabbit anti-PAK4 (1:1000; Cell Signaling Technology), mouse anti-Rac1 (1:1000; Millipore), mouse anti-GAPDH (1:50,000; Fitzgerald), mouse anti-actin (1:10,000; Sigma-Aldrich), rabbit anti-FMRP (1:1000; Abcam), rabbit anti-PSD95 (1:1000; Cell Signaling Technology), mouse anti-SV2 (1:2000; Developmental Studies Hybridoma Bank), mouse anti-VAMP2 (1:1000; Thermo Fisher Scientific), rabbit histone H3 (1:1000; Cell Signaling Technology), horseradish peroxidase (HRP)-linked rabbit anti-immunoglobulin G (IgG) (1:5000; Cell Signaling Technology), and HRP-linked mouse anti-IgG (1:5000; Cell Signaling Technology).

Pyronneau A., He Q., Hwang J.Y., Porch M., Contractor A, & Zukin R.S. (2017). Aberrant Rac1-cofilin signaling mediates defects in dendritic spines, synaptic function, and sensory perception in fragile X syndrome. Science signaling, 10(504), eaan0852.

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Detailed Reagents and Materials Protocol

Cited 4 times

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BAY-747 and runcaciguat were supplied by Bayer AG (Leverkusen, Germany)^{25 (link),26 (link)}. Donepezil, an acetylcholinesterase inhibitor (AChEI), was generously donated by Abbott (Weesp, The Netherlands). Methyl 2-hydroxyethyl cellulose (Tylose^® MH300) and Tween80 (polyoxyethylenesorbitan monooleate, cat# P8074) were purchased from Sigma-Aldrich Chemie bv (Steinheim, Germany). Forskolin was purchased from Tocris Bioscience (#1099, Abingdon, UK), rolipram was purchased from Abcam (#ab120029), and sulfo-NHS-SS-biotin (#A39258) and streptavidin-coated Dynabeads (#65601) were purchased from Thermo Scientific (Bleiswijk, The Netherlands).
Mouse anti-GluA1 was purchased from Merck Millipore (#MAB2263, Burlington, MA, USA). Rabbit anti-GluA1 phospho S845 was purchased from Abcam (#ab76321), and mouse anti-GAPDH was purchased from Fitzgerald Industries (#10R-G109A, Acton, MA, US) (all three antibodies successfully used previously^{13 (link),20 (link)}). Rabbit anti-TrkB (#4603S, Cell Signaling Technologies) and mouse anti-PSD95 (#56452, QED Bioscience) were a generous gift from Bayer (as used previously^{27 (link),28 (link)}).

Nelissen E., Possemis N., Van Goethem N.P., Schepers M., Mulder-Jongen D.A., Dietz L., Janssen W., Gerisch M., Hüser J., Sandner P., Vanmierlo T, & Prickaerts J. (2022). The sGC stimulator BAY-747 and activator runcaciguat can enhance memory in vivo via differential hippocampal plasticity mechanisms. Scientific Reports, 12, 3589.

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Western Blot Analysis of Apoptosis Markers

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Cells were lysed with RIPA buffer, and protein concentration was determined by BCA assay (Pierce, Waltham, MA). Protein lysates were separated by SDS–PAGE, transferred to nitrocellulose membranes (Bio-Rad), blocked with 5% milk/TBS, and probed with antibodies in 4°C overnight. The following antibodies were used for Western blot: mouse anti-CRT (Enzo Life Sciences, Farmingdale, NY), mouse anti-GAPDH (Fitzgerald, Acton, MA), rabbit anti-caspase 3 (Cell Signaling Technology, Danvers, MA), rabbit anti-caspase 7 (Cell Signaling Technology, Danvers, MA), rabbit anti-BAX antibody (abcam, Cambridge, MA), rabbit anti-PARP (Cell Signaling Technology, Danvers, MA), mouse anti-PARP C2-10 (Trevigen, Gaithersburg, MD), mouse anti-p53 (Santa Cruz, Dallas, TX), rabbit anti-CaMKII and anti-phospho CaMKII (Cell Signaling Technology, Danvers, MA).

Han A., Li C., Zahed T., Wong M., Smith I., Hoedel K., Green D, & Boiko A.D. (2019). Calreticulin is a Critical Cell Survival Factor in Malignant Neoplasms. PLoS Biology, 17(9), e3000402.

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Protein Extraction and Western Blot Analysis

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Protein was extracted using NP-40 lysis buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.5 mM EDTA, and 10% glycerol) containing protease inhibitors (Roche cOmplete^™, Cat # 11 697 498 001). Protein was quantitated using the BCA kit (Pierce, Cat # 23227). 20 μg of protein was resolved on a 10% SDS-PAGE gel, electroblotted onto PVDF membranes (Millipore, Immobilon-P, Cat # IPVH20200) and blocked in 5% non-fat dry milk prepared in TBS with 0.1% Tween-20 (TBST). Three washes were performed in TBST, and the following primary and secondary antibodies were used: rabbit anti-p53 (Leica Biosystems, NCL-L-p53-CM5p, 1:5000), rabbit anti-METTL3 (Abclonal, A8370, 1:1000), rabbit anti-METTL3 (Abcam, ab195352, 1:1000), mouse anti-MDM2 (Abcam, ab16895, 1:1000), mouse anti-Flag (Sigma, F1804, 1:1000), rabbit anti-HA (Thermo Fisher Scientific, 71–5500, 1:500), mouse anti-p53 (Cell Signaling Technology, 2524, 1:1000), mouse anti-GAPDH (Fitzgerald, 10R-G109A, 1:10,000), peroxidase Affinipure goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, 111–035-144, 1:5,000), and peroxidase Affinipure goat anti-mouse IgG (H+L) (Jackson ImmunoResearch, 115–035-003, 1:5,000). Immunodetection was performed using ECL^™ Prime (Millipore-Sigma, Cat# GERPN2232) or Clarity^™ Western ECL substrate (Bio-Rad, Cat# 1705060).

Raj N., Wang M., Seoane J.A., Zhao R.L., Kaiser A.M., Moonie N.A., Demeter J., Boutelle A.M., Kerr C.H., Mulligan A.S., Moffatt C., Zeng S.X., Lu H., Barna M., Curtis C., Chang H.Y., Jackson P.K, & Attardi L.D. (2022). The Mettl3 epitranscriptomic writer amplifies p53 stress responses. Molecular cell, 82(13), 2370-2384.e10.

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