Multicolor broad range protein ladder

The non-reducing gradient gel electrophoresis was performed as described (18 (link)). Membrane and cytoplasmic proteins were extracted as described in Extraction of Cell Membrane and Cytoplasmic Proteins. The protein samples were denatured with a loading buffer containing no reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol (2-ME). Samples were boiled at 100°C for 15 min. The remaining steps were essentially identical to those in Western blotting. When detecting the BCRP polymer, membrane proteins were separated by 6% Tris-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a multicolor broad range protein ladder ranging from 10 to 260 kDa (Thermo Scientific, Waltham, MA, USA) was used.

Hydrochloric acid (37%), sodium
dodecyl sulfate (98.5%), glycerol (>99%), 1,4-dithiothreitol (>98%),
bromophenol blue, tetraethyl orthosilicate (98%), fluorescein isothiocyanate
isomer (>90%), 3-aminopropyltrimethoxysilane (>97%), ammonia
(35%),
ethanol (99.8%), acetic acid (analytical standard), 2-mercaptoethanol
(>99%), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES,
99.5%), glycine (>99%), succinic anhydride (>99%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (EDC), N-hydroxysulfosuccinimide sodium
salt (>98%), NaCl (>99.5%), Tween 20, and dimethylsulfoxide
(>99.9%)
were purchased from Sigma-Aldrich and used without further purification.
Multicolor broad range protein ladder, Micro BCA assay kit and Pierce
LAL chromogenic endotoxin quantitation kit were purchased from Thermo
Fisher Scientific. NHS-PEG8-Maleimide was purchased from Irish Biotech
GMBH. Normal Human serum was purchased from Merck-Millipore Inc.