Anti dmt1

Sample containing 60-100 μg of protein was equally loaded on two parallel gels. The proteins depending upon the molecular weight were resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane (PVDF) (Bio-Rad Laboratories, Inc., Hercules, CA)as described elsewhere [26] (link). To prevent non-specific binding, PVDF membrane was incubated with 5% skimmed milk (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 60 min. Incubation of membrane was done with the primary antibodies [anti-transferrin, anti-ferroportin, anti-DMT-1, anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX), anti-ceruloplasmin or anti-hepcidin (Abcam, Cambridge, UK)] for overnight followed by 1 h incubation with anti-goat/anti-rabbit/anti-mouse (Santa Cruz Biotechnology Inc., Dallas, TX) secondary antibody conjugated with alkaline phosphatase (AP). Since the secondary antibody used in the experiment was AP-conjugated, one gel was processed for target protein and other for respective loading control. The protein bands were developed using AP specific chromogenic substrates, 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride (Thermo Fischer Scientific, Waltham, MA). Band density ratio was calculated in relation to β-actin using Alpha Imager software.