G8795

Manufactured by Merck Group

Sourced in United States

The G8795 is a laboratory equipment product manufactured by the Merck Group. It is designed to perform basic laboratory functions, but a detailed and unbiased description of its core function cannot be provided without the risk of extrapolation or interpretation.

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Lab products found in correlation

Odyssey infrared imaging system, by LI COR (10 mentions) Gapdh, by Merck Group (6 mentions) Image studio software, by LI COR (5 mentions) Protease inhibitor cocktail, by Roche (5 mentions) Odyssey clx imaging system, by LI COR (4 mentions) Trans blot turbo transfer system, by Bio-Rad (3 mentions) A2228, by Merck Group (3 mentions) F3165, by Merck Group (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Complete protease inhibitor cocktail, by Roche (3 mentions) Anti gapdh, by Merck Group (3 mentions) Ripa buffer, by Thermo Fisher Scientific (3 mentions) Ab56342, by Abcam (3 mentions) A301 985a m, by Fortis Life Sciences (3 mentions) β actin, by Merck Group (3 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Ab32064, by Merck Group (2 mentions) A21109, by Thermo Fisher Scientific (2 mentions) Polyvinylidene difluoride membrane, by Merck Group (2 mentions)

Spelling variants of this product

GAPDH (G8795) (19 citations) Anti-GAPDH (G8795) (7 citations) G8795-200UL (2 citations) Anti-GAPDH antibody (G8795) (2 citations)

86 protocols using g8795

SARS-CoV-2 Viral Protein Analysis via Western Blot

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Viral protein accumulation was analyzed by Western blot of Vero E6 cell lysates, previously transfected with 2.5 μg of circRNAs and infected with SARS-CoV-2 at an MOI of 0.1 pfu/cell at 33°C. Total protein lysates obtained at 24 h post-infection were heat-denatured in SDS-loading buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 2.5% 2-mercaptoethanol, and 0.05% bromophenol blue) at 95°C for 10 min. Following separation by SDS-polyacrylamide gel electrophoresis (PAGE; 10%), proteins were blotted onto a nitrocellulose membrane (BioRad). Viral proteins were immunostained overnight with rabbit anti-SARS nucleocapsid protein (N, 200-401-A50; Rockland Immunochemicals, 1:500) or mouse anti-GAPDH antibody (monoclonal antibody G8795, GAPDH-71.1; Sigma-Aldrich, 1:5000) and appropriate secondary antibodies (HRP-conjugated anti-rabbit (A0545-1ML) or anti-mouse (A9044-2ML), Sigma-Aldrich, each 1:10,000). The blots were developed using the Lumi-Light Western-Blotting Substrate (Roche). Western blot signals were estimated by densitometry, using GelAnalyzer 19.1 software.
For subcellular fractionation, 1.25% of cytoplasmic or nuclear fractions were analyzed by SDS-PAGE (10%) and Western blotting through detection of hnRNP A1 (monoclonal antibody, sc-32301, 4B10; Santa Cruz Biotechnology, 1:2000) and GAPDH (monoclonal antibody G8795, GAPDH-71.1; Sigma-Aldrich,1:5000).

Pfafenrot C., Schneider T., Müller C., Hung L.H., Schreiner S., Ziebuhr J, & Bindereif A. (2021). Inhibition of SARS-CoV-2 coronavirus proliferation by designer antisense-circRNAs. Nucleic Acids Research, 49(21), 12502-12516.

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Immunoblotting Analysis of PKA Subunits

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Cell extracts were prepared as described previously (Pidoux et al., 2014 (link)). Protein samples were resolved by SDS-PAGE and immunoblotted with antibodies (catalog numbers and supplier are indicated unless stated above) against PKA RIα (0.25 μg/ml; 4D7; BD Biosciences), PKA RIIα (0.25 μg/ml; 612243; BD Biosciences), PKA RIIβ (0.25 μg/ml; 610626; BD Biosciences); PKA Cα/β (0.25 μg/ml; 610980; BD Biosciences); AKAP18 (0.5 μg/ml; WH0009465M1; Sigma–Aldrich); GAPDH (1 μg/ml; G8795; Sigma–Aldrich). After incubation with the appropriate HRP-conjugated secondary antibody, blots were developed by using Supersignal West Pico substrate (Thermo Scientific, Illkirch, France).

Gerbaud P., Taskén K, & Pidoux G. (2015). Spatiotemporal regulation of cAMP signaling controls the human trophoblast fusion. Frontiers in Pharmacology, 6, 202.

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Subcellular Protein Extraction and Analysis

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Total proteins were extracted using RIPA buffer (50 mM Tris (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate) supplemental with protease inhibitors (Roche). KeyGEN Nuclear and Cytoplasmic Protein Extraction Kit (KGP150, KeyGEN BioTECH) was used to isolate nuclear proteins. Antibodies against MCM3 (ab4460, Abcam), p65 (ab16502, Abcam), p84 (ab487, Abcam), IKKβ (ab124957, Abcam), p-IKKβ (ab38515, Abcam), IκBα (ab32518, Abcam), p-IκBα (ab133462, Abcam), DNA PKcs (ab32566), DNA PKcs (phosphor S2056) (ab18192), CLEAVED PARP1 (ab32064) and GAPDH (G8795, Sigma).

Meng W., Xie B., Yang Q., Jia C., Tang H., Zhang X., Zhang Y., Zhang J., Li H, & Fu B. (2019). Minichromosome maintenance 3 promotes hepatocellular carcinoma radioresistance by activating the NF-κB pathway. Journal of Experimental & Clinical Cancer Research : CR, 38, 263.

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Quantifying Iron Homeostasis Protein Levels

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This was performed as previously described (Huebner et al. 2016 (link)). Briefly, 10 μg protein per sample was analyzed. Primary antibodies for iron homeostatic proteins were used in a 1:2000 dilution against FTN heavy chain (sc-25617, Santa Cruz Biotechnology, Dallas TX), transferrin (TF sc-30159, Santa Cruz Biotechnology), transferrin receptor-1 (TFRc 13-6890, Life Technologies, Rockville MD), and GAPDH (1:5000, G8795; Sigma-Aldrich, St Louis MO). Immunoreactive bands were visualized utilizing isotype-specific IR-dye conjugated secondary antibodies (A-21109, Life Technologies; #926-68022, 1:15,000, LI-COR, Lincoln, NB; #610-132-007, 1:30,000, Rockland Immunochemicals, Gilbertville, PA). Band IR intensities were quantified using the Odyssey infrared imaging system and Image Studio software (both from LI-COR). Molecular weights for bands of interest were calculated against color-tagged protein standards (Precision Plus Protein, BioRad, Hercules CA). Protein band intensity was normalized to GAPDH expression within the same sample and protein abundance was compared between samples; PAE does not affect GAPDH abundance in this model, as determined by comparison to total protein loaded per lane (Huebner et al. 2016 (link)). Samples were assayed in triplicate.

Huebner S.M., Helfrich K.K., Saini N., Blohowiak S.E., Cheng A.A., Kling P.J, & Smith S.M. (2018). Dietary Iron Fortification Normalizes Fetal Hematology, Hepcidin, and Iron Distribution in a Rat Model of Prenatal Alcohol Exposure. Alcoholism, clinical and experimental research, 42(6), 1022-1033.

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ELISA and Western Blot for TIMP-1

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For enzyme-linked immunosorbent assay (ELISA), platelets were homogenized in lysis buffer containing 10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.5% Triton X-100 and protease inhibitor cocktail (Sigma). Total levels of TIMP-1 were measured in lysates following the protocol for the Human TIMP-1 ELISA Kit (RAB0467, Millipore). For western blotting, the collected cells were homogenized in RIPA buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, pH 8.0, 1% Triton X-100, 0.1% SDS, and 0.5% 21-hydroxyprogesterone) containing a protease inhibitor cocktail (Sigma) and 0.5 mm sodium pervanadate. Supernatants were isolated and resolved by SDS-PAGE with reducing sample buffer before being transferred onto the appropriate membrane and incubated with antibodies. Primary antibodies for TIMP1 (WH0007076M1, Sigma-Aldrich) and GAPDH (G8795, Sigma-Aldrich), and secondary antibodies anti-mouse-HRP (A9044, Sigma-Aldrich) were used for detection. Band intensities were quantified using ImageJ software, and the results were normalized to GAPDH and expressed relative to control subjects.

Yang L., Jiang Q., Li D.Z., Zhou X., Yu D.S, & Zhong J. (2019). TIMP1 mRNA in tumor-educated platelets is diagnostic biomarker for colorectal cancer. Aging (Albany NY), 11(20), 8998-9012.

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Western Blot Protein Quantification

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Collected cells were washed with 1× PBS buffer, prepared with RIPA buffer supplemented with protease/phosphatase inhibitor cocktail, and centrifuged at 12,000 r/min for 5 min at 4°C to yield the total protein extract in the supernatants. The protein concentration was measured with a BCA assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Equal amounts of protein were separated by 8% SDS‐PAGE and subsequently transferred to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 5% nonfat milk in Tris‐buffered saline with Tween^®20 (TBST) for approximately 1 hr, followed by incubation with primary anti‐GAPDH (1:5,000; G8795, Sigma‐Aldrich), anti‐β‐actin (1:5,000; A1978, Sigma‐Aldrich), and rabbit polyclonal anti‐POU6F2 (1:1,000, TA351549, OriGene Technologies) antibodies overnight at 4°C. After washing with TBST, membranes were incubated with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1 hr. ImageJ (NIH) was used to quantify the protein band densities.

Miao Y., Li C., Guo J., Wang H., Gong L., Xie W, & Zhang Y. (2019). Identification of a novel somatic mutation of POU6F2 by whole‐genome sequencing in prolactinoma. Molecular Genetics & Genomic Medicine, 7(12), e1022.

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Western Blot Analysis of Apoptosis Markers

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Lysates were boiled for 5 minutes at 95 °C and an aliquot was resolved by SDS-PAGE and transferred to nitrocellulose by wet transfer in 10-20% methanol Tris-Glycine transfer buffer. Membranes were stained with Ponceau S Red before being dried and blocked with either 5% BSA or 5% non-fat dry milk in TBS + 0.1% Tween-20 (TBS-T) for 1 hour at room temperature. Membranes were incubated with primary antibody overnight at 4°C, washed 3 x 10 minutes with 0.1% TBS-T and secondary antibody was added for 1 hour at room temperature. Following another round of washes, proteins were visualized using enhanced chemilumescence. Antibodies were used as follows: caspase-1 (total and p20), 1:1000 (Genentech, acquired through MTA); caspase-8 (cleaved, p41/43), 1:1000 (Cell Signaling #CST8592); caspase-3 (total), 1:750 (Cell Signaling #CST9662); phospho-MLKL (Ser345), 1:1000 (Abcam #ab196436); MLKL (total), 1:2000 (Abcam #ab172868); GAPDH, 1:3000 (Sigma #G8795).

Schneider C., Shen C., Gopal A.A., Douglas T., Forestell B., Kauffman K.D., Rogers D., Artusa P., Zhang Q., Jing H., Freeman A.F., Barber D.L., King I.L., Saleh M., Wiseman P.W., Su H.C, & Mandl J.N. (2020). Migration-induced cell shattering due to DOCK8-deficiency causes a type-2 biased helper T cell response. Nature immunology, 21(12), 1528-1539.

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ROCK1/2 Expression in HUVECs under GHS Conditions

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The HUVECs on Flat, HP1, HP2, and HP3 GHS for 2 days were washed twice with PBS and disrupted with 1 × cell lysis buffer (9803, Cell Signaling Technology) containing 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma). A quantitative analysis of the samples was performed using the Bradford assay dye reagent (500–0006, Bio-Rad). The sample protein (10 μg) was boiled in 1 × loading dye for 5 min and subjected to electrophoresis in a 10% polyacrylamide gel with sodium dodecyl sulfate. After transfer to a polyvinylidene fluoride membrane (ISEQ. 00010, Millipore), the membranes were blocked with 5% bovine serum albumin containing 1 × TBST (a mixture of Tris-buffered saline and Tween 20; WH400028806, 3 M) at RT for 1 h. The membranes were incubated with anti-ROCK1 (1:1000; ab45171, Abcam), anti ROCK2 (1:1000; ab71598, Abcam), and anti-GAPDH (1:2000; G8795, Sigma) antibodies at RT for 2 h. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (1:3000; Santa Cruz) in TBST at RT for 1 h. Chemiluminescence was visualized with the ECL Plus reagent (32132, Thermo Fisher Scientific) and recorded on X-ray film.

Huang L.H., Cui L.H., Kim D.H., Joo H.J., Seo H.R., Choi S.C., Noh J.M., Lee K.B, & Hong S.J. (2019). Regulating response and leukocyte adhesion of human endothelial cell by gradient nanohole substrate. Scientific Reports, 9, 7272.

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Western Blot Analysis of BAG4 Protein

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Cells were collected and washed twice with PBS and then lysed with lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) for 30 min on ice. Xenograft tissues were ground up in liquid nitrogen and lysed with 100–200 µl lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) for 30 min on ice. The proteins were then centrifuged at 11,000 × g for 20 min at 4°C. The concentrations of proteins were detected by BCA kit (Bioworld, Guangzhou, China). Then, 30 µg protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA for 1 h and incubated with rabbit polyclonal anti-BAG4 (ab2048; 1:100; Abcam), mouse monoclonal anti-α-tubulin (T6199; 1:1,000; Sigma-Aldrich; Merck KGaA) and anti-GAPDH (G8795; 1:1,000; Sigma-Aldrich; Merck KGaA) primary antibodies for overnight at 4°C. The membranes were washed three times for 10 min with PBST (PBS 1,000:Tween-1) and incubated with HRP-conjugated goat anti-rabbit (FDR007; 1:10,000; Fdbio Science, Hangzhou, China) or anti-mouse (FDM007; 1:10,000; Fdbio Science) for 1 h at 37°C. The membranes were then washed three times for 10 min with PBST and visualized with Pico ECL (Fdbio Science) by tanon-5200 (Tanon Science and Technology Co., Ltd., Shanghai, China). α-tubulin and GAPDH served as internal controls.

Yi L., Lv Z., Wang J, & Zhong X. (2017). Bcl-2 associated athanogene 4 promotes proliferation, migration and invasion of gastric cancer cells. Molecular Medicine Reports, 16(4), 3753-3760.

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Extraction and Western Blot Analysis of Phospho-Smad Proteins

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Proteins from tail tendons were extracted from frozen tissue with ice-cold cell lysis buffer (20 mM Tris/HCl pH 7.6, 150 mM NaCl, 1 mM ethylene diamine tetra-acetic acid (EDTA), 1% (v/v) Igepal CA-630, 50 mM sodium fluoride) containing complete, mini EDTA-free protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche). Lysates were cleared by centrifugation at 10 000 × g, at 4oC. Commercially available antibodies used to detect phospho-Smad1/5 (#9516), Smad1 (#6933), Smad5 (#9517), phospho-Smad2 (#3108) and Smad2 (#3103) were purchased from Cell Signaling Technologies. GAPDH was detected using a monoclonal antibody from Sigma (G8795).

Yeung C.Y., Gossan N., Lu Y., Hughes A., Hensman J.J., Bayer M.L., Kjær M., Kadler K.E, & Meng Q.J. (2014). Gremlin-2 is a BMP antagonist that is regulated by the circadian clock. Scientific Reports, 4, 5183.

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