MISTIC-fusion proteins as antigens for high quality membrane protein antibodies.
Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this "antibody bottleneck". We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens.
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Natalia Silva
NS
Alves
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Susanne Adina
SA
Astrinidis
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Nathalie
N
Eisenhardt
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Cornelia
C
Sieverding
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Josef
J
Redolfi
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Michael
M
Lorenz
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Marion
M
Weberruss
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Daniel
D
Moreno-Andrés
Friedrich Miescher Laboratory of the Max Planck Society, Spemannstraße 39, 72076 Tübingen, Germany.
Wolfram
W
Antonin
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000941:Antigens / Q000378:metabolism*; D037281:Calnexin / Q000378:metabolism; D002846:Chromatography, Affinity; D007106:Immune Sera / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D012995:Solubility; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
D000906:Antibodies; D000941:Antigens; D007106:Immune Sera; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D037281:Calnexin