Saltar al contenido
Merck

Transmembrane protein western blotting: Impact of sample preparation on detection of SLC11A2 (DMT1) and SLC40A1 (ferroportin).

PloS one (2020-07-10)
Yoshiaki Tsuji
RESUMEN

Western blotting has been widely used for investigation of protein expression, posttranslational modifications, and interactions. 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. 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. 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. 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°C, 5min) caused the loss of resolution due to DMT1 protein aggregates. Unheated samples also resulted in better resolution for Fpn1 and TfR1 western blots. Conversely, only heated samples allowed to detect ferritin H, otherwise ferritin polymers failed to get into the gel. Neither different lysis/sample loading buffers nor sonication improved the resolution of DMT1 and Fpn1 western blots. Thus, heating samples most critically affected the outcome of western blotting, suggesting the similar cases for thousands of other transmembrane and heat-sensitive proteins.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
ANTI-FLAG® M2 monoclonal antibody produced in mouse, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Sigma-Aldrich
Ammonium iron(III) citrate, reagent grade, powder
Sigma-Aldrich
Deferoxamine mesylate salt, powder, ≥92.5% (TLC)
Sigma-Aldrich
Anticuerpo anti-gliceraldehído-3-fosfato deshidrogenasa, clon 6C5, clone 6C5, Chemicon®, from mouse
Sigma-Aldrich
Anticuerpo de cabra anti-IgG de conejo conjugado con peroxidasa, 1 mg/mL (after reconstitution), Chemicon®
Sigma-Aldrich
Anticuerpo de cabra anti-IgG de ratón, conjugado con HRP, especie adsorbida, 0.8 mg/mL, Chemicon®
Deferoxamine mesylate salt, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
MISSION® esiRNA, targeting human SLC40A1