G2781

Manufactured by Merck Group

Sourced in United States

The G2781 is a laboratory equipment product from Merck Group. It is a precision instrument designed for specialized applications in scientific research and analysis. The core function of the G2781 is to [insert concise, factual description of the product's core function without interpretation or extrapolation].

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Lab products found in correlation

Loading buffer, by Bio-Rad (1 mentions) Ab134175, by Abcam (1 mentions) Nonfat dry milk, by Cell Signaling Technology (1 mentions) 2 mercaptoethanol, by Bio-Rad (1 mentions) Trans blot turbo transfer system, by Bio-Rad (1 mentions) Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions) Immpact dab peroxidase substrate, by Vector Laboratories (1 mentions) Ab150129, by Abcam (1 mentions) Ds vi1, by Nikon (1 mentions) S3023, by Agilent Technologies (1 mentions) Ab150074, by Abcam (1 mentions) Baf 0010 03a, by CellPath (1 mentions) Humulin r insulin, by Eli Lilly (1 mentions) Non targeting shrna control, by Merck Group (1 mentions) Gapdh, by Thermo Fisher Scientific (1 mentions) Ap132p, by Merck Group (1 mentions) Srebp1, by Santa Cruz Biotechnology (1 mentions) Betulin, by Santa Cruz Biotechnology (1 mentions) L methionine sulfoximine mso, by Merck Group (1 mentions) Gapdh, by GeneTex (1 mentions)

5 protocols using g2781

Western Blot Analysis of Liver Proteins

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Snap frozen liver samples were homogenized in RIPA buffer with fresh proteinase and phosphatase inhibitor. The concentration of the protein was determined by the bicinchoninic acid assay. Protein sample was prepared with loading buffer (Bio-Rad, 1,610,737) with 5% 2-Mercaptoethanol (Bio-Rad, 161-0710) and subjected to electrophoresis. Protein sample was separated on pre-cast 7.5% or 4-20% polyacrylamide gels (Bio-Rad) and transferred to the PVDF membrane using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were stained with Ponceau-S and blocked for 30 min with 5% nonfat dry milk (Cell signaling, 9999) or 5% BSA in Blotto buffer (0.15M NaCl, 0.02M Tris pH 7.5, 0.1% Tween in dH2O), and incubated with primary antibodies at 4°C overnight at the following concentrations: GS (Sigma, G2781, 1:2000), CYP2E1 (Sigma, HPA009128, 1:1000), CYP1A2 (Santa Cruz Biotechnology, sc-53241, 1:1000), Cyclin D1 (Abcam, ab134175, 1:1000). Membranes were washed in Blotto buffer and incubated with the appropriate HRP-conjugated secondary antibody for 60 min at room temperature. Membranes were washed with Blotto buffer, and bands were developed utilizing SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, 34,080) and visualized by time-gradient autoradiography.

Hu S., Liu S., Bian Y., Poddar M., Singh S., Cao C., McGaughey J., Bell A., Blazer L.L., Adams J.J., Sidhu S.S., Angers S, & Monga S.P. (2022). Single-cell spatial transcriptomics reveals a dynamic control of metabolic zonation and liver regeneration by endothelial cell Wnt2 and Wnt9b. Cell Reports Medicine, 3(10), 100754.

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Immunohistochemical Localization of Glutamine Synthetase

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The tissue sections were deparaffinized with xylene and rehydrated through graded ethanol series and water. The sections were treated for 30 min with 0.3% hydrogen peroxide/phosphate-buffered saline (PBS) to quench endogenous peroxidase activity and then incubated for 30 min with 2.5% normal horse serum. Following 24-h incubation at 4°C with rabbit anti-GS primary antibody (#G2781, Sigma-Aldrich, St. Louis, MO, USA; 1:50,000 dilution for liver sections, 1:60,000 for brain sections), the sections were rinsed in 0.1% Tween 20/PBS and then incubated for 40 min at room temperature with horse anti-rabbit IgG peroxidase-polymer secondary antibody (ImmPRESS, Vector Laboratories, Burlingame, CA, USA). Following washing with 0.1% Tween 20/PBS, the signals were developed with diaminobenzidine (ImmPACT DAB peroxidase substrate, Vector Laboratories) for 5 min. The liver sections were counterstained with hematoxylin for 5 min. All the sections were dehydrated through graded ethanol series, cleared in xylene, and mounted. For the negative reagent control, the primary antibody was omitted. For each of the liver and brain slide sets, all the slides were processed in the same immunostaining batch.

Soontornniyomkij V., Kesby J.P., Soontornniyomkij B., Kim J.J., Kisseleva T., Achim C.L., Semenova S, & Jeste D.V. (2016). Age and High-Fat Diet Effects on Glutamine Synthetase Immunoreactivity in Liver and Hippocampus and Recognition Memory in Mice. Current aging science, 9(4), 301-309.

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Immunofluorescent Staining of Glutamine Synthetase

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Cells were washed with PBS prior to fixation in 10% formalin buffer solution (BAF-0010-03A; CellPath) for 15–20 min. Afterwards, 0.2% Triton X-100 (X100; Sigma-Aldrich) was added for 15 min. The cells were washed twice in PBS before incubated in 5% BSA for 1 hr at RT. Primary antibodies against GS (G2781; Sigma-Aldrich; 1:4000; rabbit), and either SNAP-23 (ab166808; Abcam; 1:200; goat) or SNAP-25 (ab136493; Abcam; 1:200; goat) were diluted in 1% BSA solution and incubated overnight at 4°C. The following day, cells were washed prior to incubation with secondary antibodies donkey anti-rabbit Alexa Fluor® 555 conjugate 1:500 (ab150074; Abcam) and donkey anti-goat Alexa Fluor® 488 conjugate 1:500 (ab150129; Abcam), in a 1% BSA solution, for 1.5 hrs in the dark. Subsequently, the cells were incubated with Hoechst for 10 min. at 4°C, in a concentration of 1:2000. Afterwards, the cells were washed in PBS and then milli-Q water before mounted with glass cover slips (Fluorescent mounting medium; S3023; Dako). Images were obtained using a Nikon microscope (Az100; Nikon) equipped with a fluorescent illuminator (L200/D; Prior Scientific) and a digital camera (DS-Vi1; Nikon) connected to a personal computer. Image J (public domain software) was used for further analysis.

da Silva L.B., Poulsen J.N., Arendt-Nielsen L, & Gazerani P. (2015). Botulinum neurotoxin type A modulates vesicular release of glutamate from satellite glial cells. Journal of Cellular and Molecular Medicine, 19(8), 1900-1909.

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Western Blot Analysis of Metabolic Regulators

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Antibodies used were as follows: SREBP1 (sc-13551, Santa Cruz Biotechnology, Dallas, TX, USA; 1:500 for WB), ACC (#3676, Cell Signaling Technology, Danvers, MA, USA; 1:1000 for WB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (MA5-15738, ThermoFisher Scientific, Waltham, MA, USA; 1:10,000 for WB), GS (G2781, Sigma-Aldrich, Burlington, MA, USA; 1:1000–1:10,000 for WB, and GTX630654, GeneTex, Irvine, CA, USA; 1:1000–1:10,000 for WB), Sp1 (07-645, Millipore, Burlington, MA, USA; 1:1000 for WB), O-linked N-acetylglucosamine (clone RL2) (MABS157, Millipore, Burlington, MA, USA; 1:500 for WB), horseradish peroxidase (HRP)-conjugated anti-mouse IgG (AP124P, Millipore, Burlington, MA, USA; 1:500 for SREBP1; 1:10,000 for GAPDH; 1:500–1:5000 for GS from GeneTex; 1:500 for O-linked N-acetylglucosamine), HRP-conjugated anti-rabbit IgG (AP132P, Millipore, Burlington, MA, USA; 1:1000 for ACC; 1:500–1:5000 for GS from Sigma-Aldrich; 1:500 for Sp1), and normal mouse IgG (#31903, ThermoFisher Scientific, Waltham, MA, USA). Insulin (Humulin R) was purchased from Eli Lilly and Company. L-Methionine sulfoximine (MSO) (M5379, Sigma-Aldrich, Burlington, MA, USA) was purchased from Sigma-Aldrich. Betulin (sc-234016, Santa Cruz Biotechnology, Dallas, TX, USA) was purchased from Santa Cruz Biotechnology. The shRNA targeting GS and non-targeting control shRNA were purchased from Sigma-Aldrich.

Jhu J.W., Yan J.B., Lin Z.H., Lin S.C, & Peng I.C. (2021). SREBP1-Induced Glutamine Synthetase Triggers a Feedforward Loop to Upregulate SREBP1 through Sp1 O-GlcNAcylation and Augments Lipid Droplet Formation in Cancer Cells. International Journal of Molecular Sciences, 22(18), 9814.

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Immunofluorescence Mapping of Astrocytes

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The brains were paraffin embedded and 4 µm sagittal sections were cut using a microtome (Microm HM 340E). Immunofluorescence assay was carried out in selected sections containing the hippocampus. Samples were incubated with primary antibodies, anti-GFAP (HPA056030, Sigma Aldrich Co.), and anti-glutamine synthetase (G2781, Sigma Aldrich Co.) at 1:200 overnight at 4°C. The next day, sections were washed and incubated with the secondary antibody (SAB4600310, Sigma Aldrich Co, 1:500), for 2 hr at room temperature with agitation. Subsequently, sections were washed several times, dried, and covered in the dark with Vectashield medium (Vector Laboratories, Burlingame CA, USA) containing DAPI to counterstain nuclei.

Navarro A., García M., Rodrigues A.S., Garcia P.V., Camarinho R, & Segovia Y. (2021). Reactive astrogliosis in the dentate gyrus of mice exposed to active volcanic environments. Journal of toxicology and environmental health. Part A, 84(5).

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