Goat polyclonal anti rabbit igg hrp conjugate

Cells were cultured in a 24-well plate, 6-well plate, or 100-mm dish and washed with PBS twice prior to lysis. Cells were lysed with RiPA buffer (Millipore) (with recommended EDTA-free protease inhibitor cocktail [Sigma]) on ice for 1 h, and unsolubilized material was removed by centrifugation at 3,000 g for 15 min. Protein concentration in supernatant was determined by the BCA assay, and 20 μg protein was used for further analysis. Before electrophoretic separation of proteins, each sample was combined with an equal volume of 2 x Laemmli sample containing 5% β-mercaptoethanol. Proteins were then resolved on 8% Laemmli SDS-PAGE and transferred to PVDF membrane at 90 V for 90 min. Primary antibodies used in immunoblotting were rabbit monoclonal anti-ATP7A CT77 (Hycult biotech), mouse monoclonal anti-tubulin (Sigma, T8203), mouse Anti-Na+/K+ ATPase α-1 Antibody (Millipore 06–520), and rabbit Anti-ATP7b antibody [EPR6794] (ab124973). Secondary antibodies were goat polyclonal anti-mouse IgG HRP-conjugate (Santa Cruz, SC-2005) and goat polyclonal anti-rabbit IgG HRP-conjugate (Santa Cruz, SC-2004). All antibodies were used at a dilution of 1:1,000.