Anti gapdh

Manufactured by LI COR

Sourced in United States

The Anti-GAPDH product is a primary antibody that specifically binds to the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein. GAPDH is a commonly used internal control and housekeeping gene in various biological assays. The Anti-GAPDH antibody can be used to detect and quantify GAPDH expression levels in Western blotting and other immunoassays.

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GAPDH

5 protocols using anti gapdh

Western Blot Immunodetection Protocol

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Details have been described previously59 (link). Briefly, samples were separated and transferred onto nitrocellulose membranes, blocked and incubated in succession with primary antibody overnight at 4°C and then with horseradish peroxidase-coupled secondary antibody at a dilution of 1:2000. After washing, immunoreactive bands were detected by Odyssey Infrared Imager (LICOR Bioscience, Lincoln, NE, USA).
Antibodies used were: anti-myc (9E10), anti-Human IgG (Fc specific), anti-HA (C29F4), anti-cyclin D1, anti-cyclin D3, anti-CDK4, anti-CDK6, anti-phospho-Cyclin B1 (Ser147), anti-SUSD2 rabbit polyclonal antibody, anti-β-tublin, anti-GAPDH and IRDye secondary antibodies against mouse, rabbit or goat immunoglobulin G (Li-Cor Biosciences). The anti-CSBF/C10orf99 rabbit polyclonal antibody and were made in our lab.

Pan W., Cheng Y., Zhang H., Liu B., Mo X., Li T., Li L., Cheng X., Zhang L., Ji J., Wang P, & Han W. (2014). CSBF/C10orf99, a novel potential cytokine, inhibits colon cancer cell growth through inducing G1 arrest. Scientific Reports, 4, 6812.

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Western Blot Analysis of Cell Signaling

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Cells were seeded at 4×10^5 per well in 6-well plates with appropriate growth medium and incubated at 37°C, 5% CO2 overnight, or treated with drugs for indicated period of time before harvesting. Cell lysates were prepared by cell lysis on ice with 3X RIPA buffer containing protease inhibitors (Roche, #11836170001) and phosphatase inhibitor cocktail (Sigma, #P5726). Immunoblotting of individual protein bands was performed by overnight incubating the PVDF membranes with the following primary antibodies (all purchased from Cell Signaling) diluted in 1X Odyssey Blocking Buffer (LI-COR, #927–50000): rabbit anti-phospho-ERK (#9101), mouse anti-ERK1/2 (#9107), rabbit anti-phospho-AKT (Ser473) (#4060), mouse anti-AKT (#2920), anti-mTor (#2972), anti-Rictor (#2140), anti-GAPDH (#2118) and rabbit anti-β-Actin (#4970). Fluorescent dyes-conjugated goat anti-rabbit or anti-mouse antibodies (LI-COR, #925–68071 or #925–32210) were used as secondary antibodies to visualize the protein bands with the Odyssey CLx - LI-COR Imaging System.

Zhan H., Bhattacharya S., Cai H., Iglesias P.A., Huang C.H, & Devreotes P.N. (2020). An Excitable Ras/PI3K/ERK Signaling Network Controls Migration and Oncogenic Transformation in Epithelial Cells. Developmental cell, 54(5), 608-623.e5.

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Quantitative Analysis of Synaptic Proteins

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Equal volumes of protein extracts were loaded in each well of a 4–20% Mini-PROTEAN TGX Stain-Free Protein Gel (BioRad), separated by electrophoresis, and transferred onto an FL-PVDF membrane (Millipore). Nonspecific binding sites were blocked in Intercept Blocking Buffer (LI-COR) for 1 h. Blots were probed overnight at 4°C. Bands were visualized by two-channel multiplex fluorescence (LI-COR Odyssey Fc). Quantification of western blots was performed using LI-COR Empiria Studio software. Samples were normalized using Revert Total Protein Stain (LI-COR). Antibodies used in this study were anti-PSD-95 (Antibodies Inc.), anti-synaptophysin (Abcam), anti-GAPDH (LI-COR), and anti-beta-actin (LI-COR) (Supplementary Table 3). Individuals performing the immunoblotting experiments were blinded to the research participants’ demographic, clinical, and neuropathologic characteristics.

Lilek J., Ajroud K., Feldman A.Z., Krishnamachari S., Ghourchian S., Gefen T., Spencer C.L., Kawles A., Mao Q., Tranovich J.F., Jack CR J.r., Mesulam M.M., Reichard R.R., Zhang H., Murray M.E., Knopman D., Dickson D.W., Petersen R.C., Smith B., Ashe K.H., Mielke M.M., Nelson K.M, & Flanagan M.E. (2023). Accumulation of pTau231 at the Postsynaptic Density in Early Alzheimer’s Disease. Journal of Alzheimer's Disease, 92(1), 241-260.

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Immunoblot Analysis of JNK Signaling

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Cells were transfected with constructs using Lipofectamine 2000 (Invitrogen, Waltham, MA). The cells were lysed with 1% NP40 buffer and were immunoblotted with anti-GFP (Santa Cruz), anti-JNK (CST; Cell Signaling, Danvers, MA), anti-GAPDH(CST), anti-p-JNK(CST), 1RDye® 800CW goat anti-rabbit IgG secondary antibodies (LI-COR, Cambridge, UK) and 1RDye® 680CW goat anti-mouse IgG secondary antibodies (LI-COR). Images were captured by the Odyssey® system (LI-COR). Quantification analysis is performed by using Image J.

Cao X., Tian T., Steele J.W., Cabrera R.M., Aguiar-Pulido V., Wadhwa S., Bhavani N., Bi P., Gargurevich N.H., Hoffman E.N., Cai C.Q., Marini N.J., Yang W., Shaw G.M., Ross M.E., Finnell R.H, & Lei Y. (2020). Loss of RAD9B impairs early neural development and contributes to the risk for human spina bifida. Human mutation, 41(4), 786-799.

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Protein Extraction and Detection

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Cell lysates were prepared by using a RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with Protease Inhibitor Cocktail (PIC, Sigma-Aldrich), Phenyl-methane Sulfonyl Fluoride (PMSF, Sigma-Aldrich), and Phosphatase Inhibitor Cocktail (PhoIC A and B, Sigma-Aldrich). Equal amounts of protein of each sample were analyzed using SDS-PAGE, electrophoretic transfer, immunoblotting, and chemiluminescence detection. Either 10% or 12% SDS-PAGE was used to isolate proteins with different molecular weights. The proteins were transferred to a nitrocellulose filter membrane (PALL). Antibodies and dilutions were as follows: anti-VEGFD, 1:1000; anti-PECAM-1, 1:1000; anti-LYVE1, 1:1000; anti-FLK-1, 1:1000; anti-VEGFR3, 1:1000; anti-HA, 1:1000; anti-Flag, 1:500; anti-Myc, 1:1000; anti-GAPDH, 1:5000; anti-GSNOR, 1:2000; The Odyssey Infrared Imaging System (LI-COR Bioscience, Lincoln, Nebraska) was used to detect the immunoreactive signals. Western blot bands were quantified by measuring integrated density using the Image-J software (NIH, USA). Co-immunoprecipitation was performed through the cell lysates, and the supernatants were incubated with antibody and protein A/G PLUS-Agarose (Santa Cruz) overnight at 4℃. The bound proteins were eluted in the loading buffer and subjected to the SDS-PAGE. S-nitrosylated proteins were detected by Pierce™ S-nitrosylation Western Blot Kit (Thermo).

He Q., Qu M., Shen T., Xu Y., Luo J., Tan D., Xu C., Barkat M.Q., Zeng L.H, & Wu X. (2022). Suppression of VEGFD expression by S-nitrosylation promotes the development of lung adenocarcinoma. Journal of Experimental & Clinical Cancer Research : CR, 41, 239.

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