Total cell lysates were immunoprecipitated with antibodies against IRS2, IRS1, p85 (Upstate/Millipore) or STAT6 (M-20, Santa Cruz) and Protein G-agarose beads (Life Technologies). Samples were run on a 7.5% polyacrylamide gel and transferred onto an Immobilon membrane (Millipore, Billerica, MA). The blots were blocked with BSA (Sigma) and incubated with antibodies against phosphotyrosine (PY-20, BD Biosciences) and pSTAT6 (STAT6-pTyr641, Cell Signaling Technologies, Danvers, MA) as well as total IRS2, IRS1, or p85 (Upstate/Millipore) or STAT6 (M-20, Santa Cruz). Total cell lysates were evaluated by western blotting using antibodies to pAKT-Ser473, pAkt-Thr308, Akt, phosphorylated ribosomal S6 protein (pS6235,236) or S6 (Cell Signaling Technologies, Danvers, MA). Anti-mouse or anti-rabbit horseradish peroxidase-linked secondary antibody was used (GE Healthcare). Protein bands were detected using a chemiluminescence reagent (ECL; Denville Scientific Inc., Metuchen, NJ).
Anti mouse or anti rabbit horseradish peroxidase linked secondary antibody
Manufactured by GE Healthcare
The anti-mouse or anti-rabbit horseradish peroxidase-linked secondary antibody is a laboratory instrument used to detect and quantify target proteins in various immunoassays. It is designed to bind to the primary antibody that is specific to the target protein, and the horseradish peroxidase enzyme attached to the secondary antibody can then catalyze a colorimetric or chemiluminescent reaction, allowing the visualization and quantification of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon membrane, by Merck Group (1 mentions)
Protein g agarose bead, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Mouse anti gfp clone 3e6, by Thermo Fisher Scientific (1 mentions)
Rabbit anti ha antibody, by Rockland Immunochemicals (1 mentions)
Mouse anti ha, by Merck Group (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
2 protocols using anti mouse or anti rabbit horseradish peroxidase linked secondary antibody
1
Immunoprecipitation and Western Blotting
2
Western Blot Protein Detection
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Samples were resolved on NuPAGE gels, which were transferred to nitrocellulose membranes using the iBlot Gel Transfer Device (Invitrogen). The blots were probed with primary antibody for 1 hr at room temperature, and the primary antibodies included rabbit anti-HA antibody (Rockland, 1:1000), mouse anti-HA (Millipore, 1:1000), mouse anti-dynein intermediate chain (clone 74.1, Millipore, 1:2000), rabbit anti-dynein heavy chain (clone KIAA0325; Proteintech, Chicago, IL, 1:500), and mouse anti-GFP (clone 3E6, Molecular Probes, Eugene, OR, 1:1000). The blots were then washed three times with TBST followed by incubation with 1:10,000 anti-mouse or anti-rabbit horseradish peroxidase-linked secondary antibody (GE Healthcare), anti-mouse-800 (Rockland), or anti-rabbit 680 (Molecular Probes) for 45 min at room temperature. Blots were developed either with Amersham ECL Western blotting detection reagent (GE Healthcare) or scanned using an Odyssey CLx Infrared Imaging System (LI-COR, Lincoln, NE).
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