Anti mouse igg hrp conjugated antibody from goat

Total solublized protein of Sf9 membrane preparations (see above) used in radio ligand binding assay was determined by the Bradford method according to the manufacturers’ protocol (BioRad Protein Assay; BioRad, Munich, Germany). Aliquots of homogenized membrane preparations, corresponding to 100 μg of protein, were centrifuged at 50,000 g at 4 °C for 15 min, and the pellets were re-suspended in 50 mM Tris, pH 7.4, supplemented with 1 mM EDTA and protease inhibitors (SIGMAFAST Protease Inhibitor cocktail tablets, Sigma) at a protein concentration of 1,600 μg ml⁻¹. Membrane homogenates (15 μl) were processed and subjected to immunoblotting as described previously^{38 (link)} with the following modifications: blotting onto the nitrocellulose membrane was performed at 60 mA for 60 min. Primary antibody ANTI-FLAG M1 from mouse (Sigma, order no. F3040, lot SLBK1592V) was diluted 1:500. The secondary antibody, an anti-mouse IgG HRP-conjugated antibody from goat (Sigma, order no. A0168, lot 080M4839) was diluted 1:80,000. The washing steps after incubation with the primary and the secondary antibody were 3 × 10 min each. Control experiments in the absence of the primary antibody were not performed.