Received: MaAccepted: JPublished: July 9, 2020Ĭopyright: © 2020 Yoshiaki Tsuji. PLoS ONE 15(7):Įditor: Fanis Missirlis, Cinvestav, MEXICO Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.Ĭitation: Tsuji Y (2020) Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin). Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Our results in 12 human culture cell lysates indicated that only unheated samples prior to gel loading gave rise to clear resolution of DMT1 protein, while heated samples (95☌, 5min) caused the loss of resolution due to DMT1 protein aggregates. We performed western blotting for transmembrane iron transporter proteins SLC11A2 (divalent metal transporter 1, DMT1), SLC40A1 (ferroportin 1, Fpn1), and transferrin receptor-1 (TfR1), along with cytoplasmic iron storage protein ferritin H. We show here the case that even excellent primary antibodies failed to detect a specific protein of interest due to a routine heating practice of protein samples. Because western blotting usually involves heat-denaturation of samples prior to gel loading, clarification of detailed procedures for sample preparation have been omitted or neglected in many publications. Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions.
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