A3854

Manufactured by Merck Group

Sourced in United States, Spain, Germany, Japan

The A3854 is a laboratory equipment product manufactured by Merck Group. It is a highly versatile and accurate instrument designed for specific laboratory applications. The core function of the A3854 is to perform precise measurements and analyses required in various scientific research and testing environments.

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Lab products found in correlation

Ab10983, by Abcam (5 mentions) β actin, by Merck Group (4 mentions) Pvdf membrane, by Bio-Rad (4 mentions) Image lab software, by Bio-Rad (4 mentions) Bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Protein assay, by Bio-Rad (4 mentions) Sc 7150, by Santa Cruz Biotechnology (3 mentions) Sc 2004, by Santa Cruz Biotechnology (3 mentions) Sc 2005, by Santa Cruz Biotechnology (3 mentions) Complete protease inhibitor, by Roche (3 mentions) Pvdf membrane, by Merck Group (3 mentions) Nitrocellulose membrane, by Cytiva (3 mentions) Ab68477, by Abcam (3 mentions) Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions) Cay10006421, by Cayman Chemical (2 mentions) Dc protein assay, by Bio-Rad (2 mentions) Ab87099, by Abcam (2 mentions) Hrp conjugated goat anti rabbit igg, by Bio-Rad (2 mentions) Hybond c pure nitrocellulose membrane, by Cytiva (2 mentions) X omat film, by Kodak (2 mentions)

Close spelling variants of this product

Actin-HRP (A3854) (2 citations) Anti-β-actin-HRP (A3854) (2 citations) A3854 Monoclonal Anti‐β‐Actin−Peroxidase antibody (2 citations) Anti-β-ACTIN (A3854) (13 citations) β-actin (A3854) (5 citations) Anti-β-actin antibody (A3854) (3 citations)

103 protocols using a3854

Western Blot Analysis of HIF-1α and HIF-2α

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Cells were lysed in UREA/SDS buffer, and proteins were resolved by SDS-PAGE. After transferring the proteins onto polyvinylidene difluoride membranes, proteins were detected using a rabbit polyclonal anti-HIF-1α antibody (Cay10006421, rabbit polyclonal, Cayman Chemicals) or a goat polyclonal HIF-2α antibody (AF2997, R&D Systems). We further used a rabbit monoclonal anti-DPF3 antibody (E7F7N, #82788, Cell Signaling), a mouse monoclonal anti-beta actin antibody (A3854, Sigma Aldrich), and horseradish peroxidase-conjugated secondary antibodies as applicable (Dako, Agilent Technologies).

Protze J., Naas S., Krüger R., Stöhr C., Kraus A., Grampp S., Wiesener M., Schiffer M., Hartmann A., Wullich B, & Schödel J. (2022). The renal cancer risk allele at 14q24.2 activates a novel hypoxia-inducible transcription factor-binding enhancer of DPF3 expression. The Journal of Biological Chemistry, 298(3), 101699.

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Immunoblotting Techniques for NPC2, CD1d, and NF-κB

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Cell lysates were prepared in RIPA buffer (50 mM Tris·HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% Triton X-100 plus proteinase inhibitors). Protein concentration was determined by Bradford assay, and samples containing 10–50 µg were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes. After, membranes were blocked in 8% nonfat milk in 20 mM Tris–HCl, 150 mM NaCl, and 0.05% Tween-20 for 1 h at room temperature. Antibodies were used 2 h at room temperature for immunoblotting, including anti-NPC2 (sc-33776), anti-CD1d (sc-19632), anti-CD74 (sc-47742), anti-p65-NF-κB (sc-372), anti-Lamin (sc-6215), anti-pro-saposin (Biorbyt, orb13663), or anti-β-actin-HRP (A3854, Sigma, 30 min, room temperature).

de Mingo Pulido Á., de Gregorio E., Chandra S., Colell A., Morales A., Kronenberg M, & Marí M. (2018). Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion. Frontiers in Immunology, 9, 391.

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Quantification of IKKβ Protein Levels

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500,000 cells/well of 6-well plate was plated overnight. Next day the cells were transfected with 100 nM of siGlo (fluorescently labeled negative RNAi control; GE Dharmacon, Lafayette, CO) or miR-497-5P using Lipofectamine 2000 reagent (Life Technologies). Typically, transfection rates of 80–90% were achieved. 48 hrs later the cells were lysed in RIPA buffer and protein concentrations were measured using DC Protein Assay (Bio-Rad). 30 ug of total proteins were then separated on 4–20% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred to PVDF membrane. The membrane was blocked with 5% milk and hybridized with a rabbit polyclonal anti-IKKβ antibody (sc-7607; Santa Cruz Biotechnology, Dallas, TX), or with a mouse monoclonal anti-β-actin antibody as a loading control (A3854; Sigma-Aldrich, St. Louis, MO). After subsequent probing of the membrane with goat anti-rabbit IgG-HRP (sc-2004; Santa Cruz Biotechnology) and goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology), respectively, the proteins were visualized using Western Lighting PLUS-ECL reagents (Perkin Elmer, Waltham, MA).

Mechtler P., Singhal R., Kichina J.V., Bard J.E., Buck M.J, & Kandel E.S. (2015). MicroRNA analysis suggests an additional level of feedback regulation in the NF-κB signaling cascade. Oncotarget, 6(19), 17097-17106.

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Western Blot Analysis of Cell Signaling

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For all other western blot analysis, proteins were separated on SDS polyacrylamide gels, and western blot analysis was carried out using standard procedures and the following antibodies: FlagM2 (F1804, Sigma; diluted 1:200), IQGAP1 (ab33542, Abcam, Cambridge, MA; diluted to 1 μg/ml), phospho-S6 kinase (Thr-389) (9234, Cell Signaling Technology; diluted to 1:1000), total S6 kinase (2708, Cell Signaling Technology; diluted to 1:1000), phospho-Akt (Thr-308) (4056S, Cell Signaling Technology; diluted to 1:1000), phospho-Akt (Ser-473) (4060S, Cell Signaling Technology; diluted to 1:1000), pan-Akt (4691, Cell Signaling Technology; diluted to 1:1000), peroxidase-conjugated anti-beta actin (A3854, Sigma; diluted 1:10,000), and horseradish peroxidase conjugated goat anti-mouse secondary (Jackson; diluted 1:2000). Visualization was performed using SuperSignal West Pico Chemilumnescent Substrate or SuperSignal Femto Chemiluminescent Substrate (Thermo Fisher Scientific), per the manufacturer’s instructions. Densitometry of bands was performed using a Bio Rad Molecular Imager Chemi Doc XRS+ Imaging System and ImageJ software.

Lu R., Herrera B.B., Eshleman H.D., Fu Y., Bloom A., Li Z., Sacks D.B, & Goldberg M.B. (2015). Shigella Effector OspB Activates mTORC1 in a Manner That Depends on IQGAP1 and Promotes Cell Proliferation. PLoS Pathogens, 11(10), e1005200.

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Western Blot Analysis of Cellular Proteins

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Western blot analysis was performed as previously described by our group (Cai et al., 2009). Cells were lysed and sonicated in lysis buffer. After electrophoresis, samples were transferred onto a polyvinylidene difluoride membrane (Millipore, Temecula, CA, USA), and then immunoblotted with the following antibodies: goat polyclonal anti-SIAH-1 (1:100; sc-5505; Santa Cruz Biotechnology), rabbit polyclonal anti-α-synuclein (1:1,000; 2642; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-light chain 3 (LC3) (1:1,000; ab62721; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-E1 (1:1,000; 4891S; Cell Signaling Technology), rabbit polyclonal anti-P53 (1:1,000; 2642; Cell Signaling Technology), and mouse monoclonal β-actin (1:1,000; A3854; Sigma-Aldrich, St Louis, MO, USA). Subsequently, the following horseradish peroxidase-conjugated secondary antibodies were added: polyclonal goat anti-rabbit secondary antibody for LC3, α-synuclein and E1; polyclonal donkey anti-goat secondary antibody for SIAH-1; and polyclonal goat anti-mouse secondary antibody for β-actin (1:1,000; Beyotime Biotechnology, Jiangsu, China). Images were captured using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and band intensities were calculated by densitometric analysis using Image J software (NIH, Bethesda, MD, USA).

Cai Z.L., Xu J., Xue S.R., Liu Y.Y., Zhang Y.J., Zhang X.Z., Wang X., Wu F.P, & Li X.M. (2015). The E3 ubiquitin ligase seven in absentia homolog 1 may be a potential new therapeutic target for Parkinson's disease. Neural Regeneration Research, 10(8), 1286-1291.

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Western Blot Quantification Protocol

Cited 2 times

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Western blot analysis was performed as previously described^{15 (link), 17 (link)}. Ipsi- and contralateral grey matter tissues were sampled. Protein concentration was determined by Bio-Rad protein assay kit (Hercules, CA). An equal amount of protein (50 μg) was suspended in loading buffer, denatured at 95°C for 5 minutes and loaded on an SDS-PAGE gel. After being electrophoresed and transferred onto Hybond-C pure nitrocellulose membrane (Amersham), the membrane was blocked with nonfat milk buffer for 1 hour and then incubated with the primary antibodies. The primary antibodies were polyclonal rabbit anti CD163 (Abcam, Ab87099, 1:1000), rabbit monoclonal anti hemoglobin subunit alpha (Abcam, Ab92492, 1:2000), monoclonal mouse anti-GAPDH (Fitzgerald, 10R-G109A, 1:1000000) and monoclonal mouse anti-β actin (Sigma-Aldrich, A3854, 1:100000). The membranes were then incubated with horseradish perixidase-conjugated secondary antibodies for 1 hour at room temperature. The second antibodies were HRP-conjugated goat anti-mouse IgG (1:2000, Bio-Rad) and HRP-conjugated goat anti-rabbit IgG (1:2000, Bio-Rad). Protein band densities were detected by Kodak X-OMAT film and quantified by NIH Image J. CD163 levels were expressed as the ratio of CD163/GAPDH or CD163/β actin. Hemoglobin level was expressed as the ratio of hemoglobin/GAPDH.

Liu R., Cao S., Hua Y., Keep R.F., Huang Y, & Xi G. (2017). CD163 Expression in Neurons after Experimental Intracerebral Hemorrhage. Stroke, 48(5), 1369-1375.

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PAR Detection in PARP Inhibitor Treated Cells

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LX22 cells (5 × 10⁶ cells/sample) were treated with 0.1 µM or 1 µM PARPi-FL, [¹⁸F]PARPi or olaparib in medium for 30 min at 37 °C or left untreated. Whole cell lysates were prepared by incubating cells with RIPA Buffer with Triton X (BP-140, Boston BioProducts) containing Protease Inhibitor (Complete EASYpacks, Sigma-Aldrich) for 30 min on ice, followed by 30 min centrifugation at 10,000 × g for 30 min at 4 °C. Protein concentration of lysates was determined using a Bicinchoninic acid (BCA) assay kit (#23225, Pierce) and following the manufacturers instructions. SDS gel electrophoresis and immunoblotting were carried out following standard procedures. Signal detection was carried out using chemiluminescent substrate (#34077, Thermo Scientific). Densitometric analysis of western blots was carried out using ImageJ (NIH). To detect PAR, we used a rabbit polyclonal anti-PAR polymer antibody (1:1000 dilution, #4336-BPC-100, Trevigen) followed by a goat anti-rabbit IgG-HRP secondary antibody (1:10,000 dilution, sc-2004, SantaCruz). B-actin was used as loading control (1:1000 dilution, A3854, Sigma-Aldrich) and was stained after stripping the blot for 30 min at room temperature using stripping buffer (Amresco).

Carney B., Kossatz S., Lok B.H., Schneeberger V., Gangangari K.K., Pillarsetty N.V., Weber W.A., Rudin C.M., Poirier J.T, & Reiner T. (2018). Target engagement imaging of PARP inhibitors in small-cell lung cancer. Nature Communications, 9, 176.

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Western Blotting and Immunofluorescence Antibodies

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The antibodies used in western blotting and immunofluorescence were: mouse-anti-human Lamin A/C (MAB3211, Millipore), goat-anti-mouse Lamin A/C (N-18, Santa Cruz Biotechnology), goat-anti-Lamin B1 (sc-6217, Santa Cruz Biotechnology), mouse anti-β-Actin peroxidase conjugated (A3854, Sigma), rabbit-anti-GFP antibody (ab290, Abcam), mouse-anti-Renilla Luciferase Antibody (MAB4410, Millipore).

Wu D., Yates P.A., Zhang H, & Cao K. (2016). Comparing lamin proteins post-translational relative stability using a 2A peptide-based system reveals elevated resistance of progerin to cellular degradation. Nucleus, 7(6), 585-596.

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Quantification of PARP1 Protein Expression

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PARP1 protein expression was measured in ESO51, OE19, SKGT4 and OE33 cell lysates using Western blot analysis as described before^{56 (link)}. Briefly, proteins were isolated from cells and 30 μg of protein per sample were separated with SDS/PAGE gel electrophoresis and transferred to a Nitrocellulose membrane. Proteins were detected using antibodies specific for PARP1 (1:1000; sc-7150, Santa Cruz) and b-actin (1:2000; A3854, Sigma-Aldrich) with a corresponding horseradish peroxidase (HRP) conjugated secondary antibody (1:10,000, sc-2004, Santa-Cruz). Detection was performed using a chemiluminescent substrate (Thermo Scientific #34077, SuperSignal West Pico). Since distribution of the primary anti-PARP1 antibody was discontinued, we tested and validated PA5–16452 (Invitrogen) as potential alternative.

Kossatz S., Pirovano G., De Souza França P.D., Strome A.L., Sunny S.P., Zanoni D.K., Mauguen A., Carney B., Brand C., Shah V., Ramanajinappa R.D., Hedne N., Birur P., Sihag S., Ghossein R.A., Gönen M., Strome M., Suresh A., Molena D., Ganly I., Kuriakose M.A., Patel S.G, & Reiner T. (2020). Validation of the use of a fluorescent PARP1 inhibitor for the detection of oral, oropharyngeal and oesophageal epithelial cancers. Nature biomedical engineering, 4(3), 272-285.

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Mitochondrial Protein Characterization

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All the chemicals were obtained from Sigma-Aldrich unless otherwise specified. Following antibodies were used in this study: UCP1 antibody (ab-10983, Abcam), BCAT1 antibody (TA504360, OriGene), BCAT2 antibody (9432, Cell Signaling Tech), BCKDHA antibody (sc-271538, Santa Cruz), TOM20 antibody (11802–1-AP, Proteintech), COX-IV antibody (4850, Cell Signaling), OXPHOS cocktail (Abcam, ab110413), PDH-E1α antibody (sc-377092, Santa Cruz), PDH-E1α (pSer232) antibody (AP1063, Millipore), PDH-E1α (pSer293) antibody (ab177461, Abcam), PDH-E1α (pSer300) antibody (AP1064, Millipore), GAPDH antibody (sc-32233, Santa Cruz), and β-actin antibody (A3854, Sigma-Aldrich). Polyclonal antibody for SLC25A44 was generated by using amino acids (MEDKRNIQIIEWEHLDKKKC, MMQRKGEKMGRFQVC, and CKKLSLRPELVDSRH) as epitopes for immunization in rabbit (GeneScript).

Yoneshiro T., Wang Q., Tajima K., Matsushita M., Maki H., Igarashi K., Dai Z., White P.J., McGarrah R.W., Ilkayeva O.R., Deleye Y., Oguri Y., Kuroda M., Ikeda K., Li H., Ueno A., Ohishi M., Ishikawa T., Kim K., Chen Y., Sponton C.H., Pradhan R.N., Majd H., Greiner V.J., Yoneshiro M., Brown Z., Chondronikola M., Takahashi H., Goto T., Kawada T., Sidossis L., Szoka F.C., McManus M.T., Saito M., Soga T, & Kajimura S. (2019). BCAA catabolism in brown fat controls energy homeostasis through SLC25A44. Nature, 572(7771), 614-619.

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