Using WB, the primary antibody, rabbit polyclonal anti-MX1 antibody (ab95926, Abcam, UK), was validated. BC cell line lysate, MCF7, and human embryonic kidney (HEK) that was used as a control (from the American Type Culture Collection, Rockville, MD, USA) were employed for WB antibody specificity validation. MX1 antibody was used at a dilution of 1:1500 and IRDye 800CW Donkey anti-Rabbit fluorescent secondary antibody (LI-COR Biosciences) was used at a 1:15,000 dilution. For loading control, mouse monoclonal anti-β-actin primary antibody (1:5000, Sigma-Aldrich) was used and followed by incubation with anti-Mouse fluorescent secondary antibody (LI-COR Biosciences). To detect the protein molecular weight, 20 µg of the cell lysate was loaded alongside the protein ladder (Page Ruler Plus Prestained Protein Ladder, Thermo Scientific). A specific band was detected at the predicted molecular weight of ~ 64 kDa using Odyssey Fc scanner and visualised by Image Studio 4.0 software (Supplementary Fig. 1).
Irdye 800cw donkey anti rabbit fluorescent secondary antibody
Manufactured by LI COR
The IRDye 800CW Donkey anti-Rabbit fluorescent secondary antibody is a detection reagent used in immunoassays and Western blotting applications. It is designed to bind to rabbit primary antibodies, allowing for the visualization and quantification of target proteins. The antibody is conjugated with the IRDye 800CW fluorescent dye, which can be detected using near-infrared imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (2 mentions)
Mcf 7, by American Type Culture Collection (2 mentions)
Mouse monoclonal anti β actin primary antibody, by Merck Group (1 mentions)
Ab95926, by Abcam (1 mentions)
A5441, by Merck Group (1 mentions)
Ab168352, by Abcam (1 mentions)
2 protocols using irdye 800cw donkey anti rabbit fluorescent secondary antibody
1
Validating MX1 Antibody Specificity by Western Blotting
2
Validating GLUD1 Antibody Specificity
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GLUD1 primary antibody specificity (Rabbit monoclonal, Ab168352, Abcam Plc, Cambridge UK) was determined using western blotting with MCF7 and MDA-MB-231 human BC cell lines (obtained from the American Type Culture Collection; Rockville, MD, USA). GLUD1 primary antibody was used at a 1:250 dilution and IRDye 800CW Donkey anti-Rabbit fluorescent secondary antibody (926-32213, LI-COR Biosciences) was used at a 1:15000 dilution. Mouse monoclonal anti-β-actin primary antibody (1:5000, A5441, Sigma-Aldrich) with IRDye 680RD Donkey anti-Mouse fluorescent secondary antibody (1;15000, 926-68072, LI-COR Biosciences) was used as a control. Samples were loaded at 10µg alongside the protein ladder (26619, PageRuler Plus Prestained Protein Ladder, Thermo Scientific) to determine the correct molecular weight. Odyssey Fc with Image Studio 4.0 was used to visualise protein bands (LI-COR Biosciences) which showed a single specific band at the predicted molecular weight of 62 KDa (Figure 1A).
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