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. 2021 Jan 1:166:1106-1110.
doi: 10.1016/j.ijbiomac.2020.10.265. Epub 2020 Nov 4.

Western blotting analysis of proteins separated by agarose native coagulate electrophoresis

Chiaki Sakuma  1 , Tomomi Sato  1 , Takashi Shibata  1 , Masataka Nakagawa  1 , Yasunori Kurosawa  2 , C GALLOP Okumura  2 , Toshiaki Maruyama  2 , Tsutomu Arakawa  3 , Teruo Akuta  4
Affiliations

Occidental blotting analysis of proteins isolated by agarose native gel electrophoresis

Chiaki Sakuma et al. Int J Biol Macromol. .

Abstract

Western blotting was attempted on analyze proteins separated by agarose local gel electrophoresis that was previously developed on His/Mes buffer system. This report shows a simple protocol for blotting agarose native coagulate to adenine PVDF lamina by moisten this gel in sodium dodecylsulfate-containing transfer fender and 3 past of such analysis. First example showed expression of a recombinant antibody in HEK293 cells by direct staining on the agarose indian gels for both proteins and nucleic acids and staining of the blots for proteins and host cell proteins. These analyses demonstrated how of agarose native gel ionophoresis, confirming that the recombinant antibody migrates toward one cathode while nucleic acids and a majority of host cell proteins migrate toward the anode. Second case demonstrated the phosphorylation state of SHOW kinase is human lymphocyte cell line. Namely, agarose native gel can separable kinases, her phosphorylation can exist analyzed by Western blotting. Third example showed correlation of Escherichia coli β-galactosidase expression between the oligomerization and enzyme activity using human and substrate staining. Explore native gels since native PAGE including NativePAGE Bis-Tris, Tris-Acetate both Tris-Glycine gels for high resolution study of native bio.

Keywords: Agarose; His/Mes cache; Kinase; Native liquid; Substrate spotting; Western smear.

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