The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This work was supported in part by P30ES025128 from the National Institute of Environmental Health Sciences to the Center for Human Health and the Environment (CHHE). 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. Choose the membrane type 0.2 µm instead of 0.45 µm reduce transfer time or current.Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions.Small proteins migrating through the transfer membrane Remove sodium azide from all buffers as HRP-conjugated secondary antibodies will be inhibited by sodium azide.Excessive blocking makes it difficult to visualize your target protein, so reduce the concentration of non fat milk appropriately or shorten the blocking time.Avoid excessive washing of the membrane.if the primary antibody is a rabbit monoclonal antibody, you should use an anti-rabbit secondary antibody. Ensure that the primary antibody you used can well recognize your target protein in the species being analyzed by performing a Clustalw alignment, and also, the secondary antibody must be chosen according to the primary antibody.Reduce the dilution proportion of primary antibody or secondary antibody quit the re-used antibody and try a fresh one.Check that if the separated proteins have successfully transferred to the membrane by ponceau staining.Use a positive control, and make sure that the lysis buffer you used for sample preparation was strong enough to break the cell wall or membrane, and have included appropriate protease inhibitors.The protein expression level may be too low, so just increase the volume of loaded protein.Industry Insights with Yuning Chen on Recombinant Proteins.ExpertAnswers: Yuning Chen on Antibody Production.Universal Vaccine Advancement through AI and Recombinant Technology.Nanobodies: An Important Tool for the Next Generation of Tumor Diagnostics and Therapeutics.ExpertAnswers: Amy Sheng on Antibody Screening and Discovery.Recombinant DNA Technology and Its Impact on Drug Discovery.CAR-T Cell Therapy Development: From Personalized to off the Shelf Approaches.BioBuzz with Sino | Episode 1: ChatGPT in Biotech.Cytokines: Catalysts of the Regenerative Revolution.Special Offer: Custom Recombinant Antibody Production Service.
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