Western blotting reagents

Manufactured by Thermo Fisher Scientific

Sourced in United States

Western blotting reagents are a set of laboratory products used in the process of Western blotting, a widely used analytical technique in molecular biology and biochemistry. These reagents facilitate the detection and quantification of specific proteins within a complex sample. The core function of Western blotting reagents is to enable the separation, transfer, and detection of proteins, allowing researchers to study their expression, abundance, and interactions.

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Lab products found in correlation

Chemidoc mp, by Bio-Rad (1 mentions) Gata1, by Abcam (1 mentions) Alpha tubulin, by Abcam (1 mentions) Acetylated tubulin, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor, by Merck Group (1 mentions) M per, by Thermo Fisher Scientific (1 mentions) Na3vo4, by Thermo Fisher Scientific (1 mentions) Glyceraldehyde 3 phosphate dehydrogenase, by Cell Signaling Technology (1 mentions) Anti phosphorylated ampkα, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Pierce™ ECL Advance Western Blotting Detection Reagents (5 citations) Enhanced chemiluminescence (ECL) Western blotting reagents (4 citations) Thermo Scientific Pierce ECL Western Blotting Detection reagents (3 citations) ECL femto Western blotting detection reagents (2 citations) Enhanced chemiluminescence western blotting reagents (2 citations) NovexR ECL Western blotting detection reagents (2 citations) ECL western blotting detection reagents and analysis system (2 citations) ECL advance Western blotting detection reagents (2 citations) Reagents for real time RT-PCR and Western blotting (2 citations) ECL western blotting detection system reagents and Films (CL-X-posure) (2 citations)

2 protocols using western blotting reagents

Western Blotting Techniques for Protein Analysis

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Western blotting reagents were purchased from Thermo Fisher Scientific unless otherwise stated. Cells were lysed in M-Per mammalian protein extraction reagent (cat#78501) supplemented with Halt® protease and phosphatase inhibitor cocktail, following manufacturer’s instructions. Quantification of total protein was achieved by using a bicinchoninic acid (BCA) assay. For each sample, 20–40μg of protein were separated by electrophoresis using 12% Tris-Glycine gels. Protein transfers were performed using the iBlot2 system (20V for 1 minute, 23V for 3 minutes, and 25V for 2 minutes setting). PVDF membranes were blocked in 4% milk- Tris Buffered Saline with Tween 20 (TBST) buffer for 1 h, incubated in primary antibody overnight followed by the HRP secondary for 1 h.
The sources of primary antibodies used were: GATA-1 (Abcam; cat#89505, mouse monoclonal), acetylated tubulin (Santa Cruz, Dallas, TX; cat#sc-23950, mouse monoclonal), alpha tubulin (Abcam; cat#4074, rabbit polyclonal), histone H3 (ProSci, Poway, CA; cat#31007), and acetylated histone H3 (Thermo Fisher Scientific; Lys6, cat#MA5–11195 and Lys36, cat#MA-24672). Secondary HRP labeled, anti-mouse (cat#6728) and anti-rabbit (cat#6721) antibodies were purchase from Abcam. Membranes were incubated for 1 min in ECL reagent and captured using ChemiDoc MP (BioRad, Hercules, CA).

Simic D, & Sang N. (2019). Compounds targeting Class II histone deacetylases do not cause panHDACi associated impairment of megakaryocyte differentiation. Experimental hematology, 72, 36-46.

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AMPK Activation and Quantification in Cultured Preadipocytes

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Cultured preadipocytes were isolated using ice-cold M-PER (Thermo Fisher Scientific, USA), protease inhibitor (Sigma-Aldrich, USA), and 2 mM Na₃VO₄ (Thermo Fisher Scientific, USA). Homogenated cells were then mixed with an equal volume of 2×standard sodium dodecyl sulfate (SDS) sample loading buffer (Invitrogen, Waltham, MA, USA). Gradient gels were used for SDS-polyacrylamide gel electrophoresis separation of proteins. Membranes were then incubated overnight at 4°C in primary antibodies: anti-AMPKα, rabbit polyclonal (Cell signaling, Danvers, MA, USA) with dilution of 1:1,000, anti-phosphorylated AMPKα, rabbit polyclonal (Cell signaling, USA) with dilution of 1:1,000. Membranes were then incubated with a secondary antibody, Alexa-Fluor 633, goat anti-rabbit, dilution at 1:2,000 dilution for 2 h at the room temperature. After three 10 min washes, membranes were visualized using enhanced chemiluminescent substrate, Western blotting reagents (Thermo Fisher Scientific, USA), and exposure to film (MR, Kodak, Rochester, NY, USA). Density of the bands were quantified using Imager Scanner II and Image Quant TL software. To reduce the variation between blots, tissue lysates of both groups were run in a single gel. Band density was normalized according to the glyceraldehyde 3-phosphate dehydrogenase (Cell signaling, USA) content within each sample.

Kim J., Chung K, & Johnson B.J. (2019). Chromium acetate stimulates adipogenesis through regulation of gene expression and phosphorylation of adenosine monophosphate-activated protein kinase in bovine intramuscular or subcutaneous adipocytes. Asian-Australasian Journal of Animal Sciences, 33(4), 651-661.

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