Goat anti mouse igg h l hrp conjugate

Total protein from the cells was extracted by the addition of TE sample buffer (0.1 M TRIS-PO₄, 4% SDS, 4 mM Na-EDTA, 40% glycerol, 0.04% bromophenol blue, and 0.04% β-mercaptoethanol; materials from Sigma-Aldrich, Burlington, MA, USA). Equal amounts of the protein samples were loaded on 10% acrylamide gels. Blots were probed with the following primary antibodies: anti-ABCG2 (BXP-21, Abcam, Cambridge, UK, cat. ab3380) and anti-GFP (Abcam, Cambridge, UK, cat. ab290). Goat anti-mouse IgG (H+L) HRP conjugate (Abcam, cat. ab97023) and goat anti-rabbit IgG (H+L) HRP conjugate (Abcam, Cambridge, UK, cat. ab6721) secondary antibodies were used to visualize and quantify the results. Detection was performed with Clarity Western ECL Substrate (BioRad, Hercules, CA, USA, cat. 1705060) and luminescence was detected with the BioRad ChemiDoc MP Imaging System. Densitometry analysis was performed by ImageJ software v1.42q.

Proteins were isolated from HEK293, HEL92 and K562 cells in TE sample buffer (0.1 M TRIS-PO4, 4% SDS, 4 mM Na-EDTA, 40% glycerol, 0.04% β-mercaptoethanol and 0.04% bromophenol blue) for Western blot experiments. For detecting the respective proteins, the anti-GATA1 (Abcam ab181544) rabbit monoclonal and anti-β-actin (Sigma, cat. A1978, St. Louis, MO, USA) mouse monoclonal primary antibodies, goat anti-mouse IgG (H + L) HRP conjugate (Abcam ab97023, Cambridge, UK) and goat anti-rabbit IgG (H + L) HRP conjugate (Abcam ab6721) secondary antibodies and Pierce™ ECL Western Blotting Substrate were used.

Immunoblotting was performed according to standard protocols. Total protein from the cells was precipitated by the addition of a sample buffer containing 0.1 M TRIS-PO₄, 4% SDS, 4 mM Na-EDTA, 40% glycerol, 0.04% bromphenol blue, and 0.04% β-mercaptoetanol (materials from Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Roche, cat. 11836153001). Red blood cell membrane proteins were prepared according to the protocol described previously^{19 (link),56 (link),57 (link)}. Proteins were TCA precipitated, washed twice with cold acetone, and resuspended in an SSP buffer (62.5 mM Tris-HCl pH6.8, 2% SDS, 10 mM EDTA-Na, pH = 6.8, 10% glycerol, 0.125 g/ml urea, 0.14 g/ml bromphenol blue, all components from Sigma Aldrich). Equal amounts of proteins were loaded on 10% acrylamide gels. Blots were probed with the following primary antibodies: anti-ABCG2 (BXP-21, Abcam, cat. ab3380), anti-GFP (Abcam, cat. ab290), and anti-β-actin (Sigma, cat. A1978). Goat anti-mouse IgG (H + L) HRP conjugate (Abcam, cat. ab97023) and goat anti-rabbit IgG (H + L) HRP conjugate (Abcam, cat. ab6721) secondary antibodies were used to visualize and quantify the results. Detection was performed with Imobilon Western Chemiluminescent HRP Substrate (Millipore, cat. WBKLS0500) and luminography. Densitometric analysis was performed by ImageJ software v1.42q.