A8275

Manufactured by Merck Group

Sourced in Germany

A8275 is a laboratory equipment product designed for general use in scientific and research environments. It serves as a device for performing various laboratory tasks and analyses. The core function of A8275 is to facilitate essential laboratory procedures, without interpretation or extrapolation on its intended use.

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Lab products found in correlation

4 protocols using a8275

Western Blotting Antibody Panel

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Primary antibodies used for western blotting were anti CBP (Anti-Calmodulin Binding Protein; DAM1411288; Millipore) used at 1∶5000, anti HA (Anti influenza hemagglutinin; H3663; Sigma) used at 1∶5000, anti CTD which recognizes Rpb1 and will be referred to in the text as anti Rpb1 (ab5408; Abcam) used at 1∶500, or finally anti PAP (Peroxidase-Anti-Peroxidase; P1291; Sigma) used at 1∶10000 and rabbit polyclonal anti Ccr4 which was generated in our laboratory and used at 1∶5000. The secondary antibodies were anti-Mouse-HRP (IgG-Peroxidase conjugate; A9044; Sigma) used at 1∶10000 or anti-Rabbit-HRP (IgG-Peroxidase conjugate; A8275; Sigma) used at 1∶10000. For detection of Rpb1, ovaries were incubated with 7G5 mouse monoclonal antibody used at 1∶1000 (a kind gift of Dr. László Tora, Strasbourg).

Villanyi Z., Ribaud V., Kassem S., Panasenko O.O., Pahi Z., Gupta I., Steinmetz L., Boros I, & Collart M.A. (2014). The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation. PLoS Genetics, 10(10), e1004569.

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Western Blot Analysis of ERα, PR, and Vinculin

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Cells were lysed and total protein was collected using NETN buffer (20 mM Tris pH 8.0, 150 nM NaCl, 1 mM EDTA and 0.5% NP40) supplemented with an EDTA-free protease inhibitor cocktail tablet (Roche, Indianapolis, IN). Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA), and equal amounts of protein lysate were suspended in Laemmli buffer (BioRad), run on 7.5% SDS-PAGE gels, and transferred onto PVDF membranes (BioRad). Membranes were blocked in 5% milk in TBST for 1 hour at room temperature, then probed for ERα (F-10, Santa Cruz Biotechnology, Inc., Dallas, TX), progesterone receptor (HC-190, Santa Cruz Biotechnology), and vinculin (EPR-8185, Abcam, Cambridge, United Kingdom). All primary antibodies were diluted in 5% milk in TBST at 1:500 (ERα), 1:1000 (PR), or 1:2500 (vinculin) and applied to the membranes at 4°C overnight. After washing, membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (Sigma Aldrich, A8275 (RRID:AB_258382) and A4416 (RRID:AB_258167), respectively) diluted 1:2000 in 5% milk in TBST for one hour at room temperature. Visualization was performed using enhanced chemiluminescence on the LI-COR Odyssey Fc system (Lincoln, NE).

Jones C.J., Subramaniam M., Emch M.J., Bruinsma E.S., Ingle J.N., Goetz M.P, & Hawse J.R. (2021). Development and characterization of novel endoxifen-resistant breast cancer cell lines highlight numerous differences from tamoxifen-resistant models. Molecular cancer research : MCR, 19(6), 1026-1039.

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Western Blot Protein Analysis

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Protein samples were prepared by boiling the pelleted cells in radioimmunoprecipitation assay buffer, as described (41 (link)). The membranes were probed with 1:5000 mouse monoclonal anti-HA (16B12; Covance), 1:5000 rabbit polyclonal anti-FRB (from David Shore, University of Geneva), 1:5000 rabbit polyclonal anti-RAP1 (from David Shore, University of Geneva), 1:10,000 rabbit polyclonal anti–glyceraldehyde-3-phosphate dehydrogenase (A9521; Sigma-Aldrich), and 1:5000 mouse monoclonal anti-GFP antibodies (11814460001, Roche). Horseradish peroxidase–conjugated goat anti-mouse (Bio-Rad) or anti-rabbit (A8275, Sigma-Aldrich) immunoglobulin G were used at 1:10,000 as secondary antibodies.

Kassem S., Ferrari P., Hughes A.L., Soudet J., Rando O.J, & Strubin M. (2020). Histone exchange is associated with activator function at transcribed promoters and with repression at histone loci. Science Advances, 6(36), eabb0333.

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Immunofluorescence and Western Blot Protocols

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The following antibodies were used for immunofluorescence: Primary antibodies anti-FAK, rabbit polyclonal 1:150 (Cat.#06-543, Upstate Biotechnology, Lake Placid, NY, USA) and anti-paxillin, clone 5H11, mouse monoclonal 1:150 (Cat.#05-417, Upstate Biotechnology, Lake Placid, NY, USA); secondary antibodies Alexa Fluor 488, goat anti-mouse 1:200 (Cat.#A11029, ThermoFisher Scientific, Waltham, MA, USA) and Alexa Fluor 488, goat anti-rabbit 1:200 (Cat.#A11034, ThermoFisher Scientific, Waltham, MA, USA). Rhodamine Phalloidin R415 (ThermoFisher Scientific, Waltham, MA, USA) 1:300 was used for visualization of actin.
The western blot technique was performed with the following antibodies: anti-FAK, clone 4.47, mouse monoclonal 1:1000 (Cat.#05-537, MERCK-Millipore, Darmstadt, Germany); anti-Rack1 mouse monoclonal, clone B3, 1:1000 (Cat.#sc-17754, Santa-Cruz Biotechnology, Dallas, TX, USA); anti-RSK (ribosomal protein S6 kinase) mouse monoclonal, 1:2000 (Cat.#610225, BD Transduction Laboratories, San Francisco, CA, USA); secondary antibodies HRP-conjugated goat anti-mouse IgG 1:80.000 (A9044, Sigma-Aldrich/Merck, Darmstadt, Germany) and goat anti-rabbit 1:5.000 (A8275, Sigma-Aldrich/Merck, Darmstadt, Germany).

Ramaniuk O., Klímová Z., Groušl T, & Vomastek T. (2020). Quantitative Phase Imaging of Spreading Fibroblasts Identifies the Role of Focal Adhesion Kinase in the Stabilization of the Cell Rear. Biomolecules, 10(8), 1089.

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