Non-specific Detection of a Major Western Blotting Band in Human Brain Homogenates by a Multitude of Amyloid Precursor Protein Antibodies

Hazal Haytural, Jolanta L. Lundgren, Tansu B. Köse, Tomàs Jordà-Siquier, Marinela Kalcheva, Mohammed Seed Ahmed, Bengt Winblad, Erik Sundström, Gaël Barthet, Lars O. Tjernberg, Susanne Frykman
Front. Aging Neurosci.. 2019-10-09; 11:
DOI: 10.3389/fnagi.2019.00273

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Haytural H(1), Lundgren JL(1), Köse TB(1), Jordà-Siquier T(2), Kalcheva M(1), Seed Ahmed M(1)(3), Winblad B(1), Sundström E(1), Barthet G(2), Tjernberg LO(1),
Frykman S(1).

Author information:
(1)Division of Neurogeriatrics, Center for Alzheimer Research, Department of Neurobiology, Care Science and Society, Karolinska Institutet, Solna, Sweden.
(2)Interdisciplinary Institute of Neuroscience, Université de Bordeaux, Bordeaux, France.
(3)Wolfson Centre for Age-Related Diseases, King’s College London, London, United Kingdom.

The use of human post-mortem brain material is of great value when investigating
which pathological mechanisms occur in human brain, and to avoid translational
problems which have for example been evident when translating animal research
into Alzheimer disease (AD) clinical trials. The amyloid β (Aβ)-peptide, its
amyloid precursor protein (APP) and the intermediate APP-c-terminal fragments
(APP-CTFs) are all important players in AD pathogenesis. In order to elucidate
which APP CTF that are the most common in brain tissue of different species and
developmental stages, and whether there are any differences in these fragments
between AD and control brain, we investigated the occurrence of these fragments
using different APP c-terminal antibodies. We noticed that whereas the
conventional APP-CTFα and CTFβ fragments were most prominent in rat and mouse
brain tissue, the major western blotting band detected in human, macaque and
guinea pig was of approximately 20 kDa in size, possibly corresponding to the
newly discovered APP-CTFη. However, this band was also intensely stained with a
total protein stain, as well as by several other antibodies. The staining
intensity of the 20 kDa band by the APP antibodies varied considerably between
samples and correlated with the staining intensity of this band by the total
protein stain. This could potentially be due to non-specific binding of the
antibodies to another protein of this size. In-gel digestion and mass
spectrometry confirmed that small amounts of APP were present in this band, but
many other proteins were identified as well. The major hit of the mass
spectrometry analysis was myelin basic protein (MBP) and a myelin removal
protocol removed proportionally more of the 20 kDa APP band than the full-length
APP and APP-CTFα/β bands. However, the signal could not be immunodepleted with an
MBP antibody. In summary, we report on a potentially non-specific western
blotting band of approximately 20 kDa and call for precaution when analyzing
proteins of this size in human brain tissue.

Copyright © 2019 Haytural, Lundgren, Köse, Jordà-Siquier, Kalcheva, Seed Ahmed,
Winblad, Sundström, Barthet, Tjernberg and Frykman.


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