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

Abstract

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.

Keywords: amyloid precursor protein; cross-reactivity; human brain; western blotting; η-secretase.

Figure 1

Elucidating the pattern of bands detected by different amyloid precursor protein (APP) antibodies in rat and human brain. (A) The APP protein, indicating the epitopes of the antibodies used in the study. (B–F) Equal amounts of homogenates of human Alzheimer disease (AD) and adult rat brain were loaded on 4%–12% (upper panels) or 16% (lower panels) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and subjected to western blotting using the Odyssey digital fluorescent system with detection by primary antibodies (B) Y188 and C1/6.1, (C) Y188 and 6E10, (D) A8717 and C1/6.1, (E) 9478 and C1/6.1 or (F) Y188 and 7N22. A recombinant C99-FLAG peptide was loaded on the 4%–12% gels to indicate the approximate position of c-terminal fragments (CTF)-β (minus the 1 kDa FLAG-tag). The figures are representative figures and four additional rat brain samples and five additional human brain samples showed a similar pattern using the Y188 and C1/6.1 antibodies (see also Figure 5). (G) Equal amounts of homogenates of human AD and adult rat brain were loaded on a 4%–12% SDS-PAGE gel and subjected to western blotting using the Odyssey digital fluorescent system with detection by primary antibody Y188 together with a total protein stain. A major band was detected around 20 kDa by all antibodies as well as by the total protein stain.

Figure 2

Bands detected by APP antibodies in different species and developmental stages. (A) Equal amounts of human AD, macaque, guinea pig, rat and mouse brain homogenates were loaded on a 16% gel and subjected to western blotting using the Y188 and the C1/6.1 antibodies. The major 20 kDa band is detected in human, macaque and guinea pig brain, but not in rat or mouse brain. (B) Equal amounts of brain homogenates from mouse embryo, adult mouse, human embryo and human adult control were loaded on a 16% gel. The Y188 and C1/6.1 antibodies were used for detection. The 20 kDa band was only detected in adult human brain whereas only lower molecular CTFs were present in human fetal brain. The pattern of CTFs was similar in embryonic and adult mouse brain. Panel (B) is a representative figure and the results were confirmed using an additional three human and four mouse embryonic brain samples.

Figure 3

Bands detected by APP antibodies in human AD and control brain homogenates. Equal amounts of homogenates form AD and control brain were loaded on 16% gels. In order to assure linearity of the signals, three different concentrations of a standard sample (A1) was loaded on all gels and a standard curve was created which was used for quantification. The gels were subjected to SDS-PAGE and western blotting using (A) Y188 antibody together with a total protein stain or (B) C1/6.1 antibody together with a total protein stain. Representative gels are shown with age- and gender-matched pairs of AD and control cases loaded beside each other. (C,D) Densitometric analysis of the 20 kDa band using the Y188 (C) or the C1/6.1 antibody (D). All values were normalized to the total protein stain and expressed as % of the standard sample. The major 20 kDa APP band varies considerably between cases and correlates with the size and intensity of a major total protein stained band. No difference in the levels of the 20 kDa band could be detected between AD and control brain. A, AD; C; control.

Figure 4

Mass spectrometry analysis of the 20 kDa band. Human AD brain homogenate was loaded on a 4%–12% gel and bands corresponding to the size of FL-APP or the 20 kDa band were cut out, in-gel digested with trypsin and subjected to liquid chromatography tandem mass spectrometry. The digested bands of both FL-APP (upper panel) and the 20 kDa band (lower panel) gave rise to spectra corresponding to the peptide LVFFAEDVGSNK of the APP sequence. Many other proteins were identified in the 20 kDa band to a higher score (Supplementary File S1).

Figure 5

Immunoprecipitation of APP and depletion of myelin and myelin basic protein (MBP). (A) Western blot of immunoprecipitation of human AD brain homogenate using the Y188, the A8717 antibody or a rabbit immunoglobulin antibody (rIgG) as negative control. A recombinant C99-FLAG peptide was loaded as a size indicator. The 20 kDa band could not be immunoprecipitated to a higher level with the specific antibodies Y188 and A8717 than with the control rIgG, and the majority of the 20 kDa remained in the unbound fraction. On the contrary, full length (FL) APP and some of the lower molecular weight CTFs, were successfully immunoprecipitated. Fifteen percentage of the brain homogenate was collected before immunoprecipitation and loaded as input. This is a representative figure of eight experiments. (B) Western blotting of a human brain AD homogenate using the MBP and the C1/6.1 antibody shows overlap of the signal in the 20 kDa band. (C) A human brain AD homogenate was subjected to myelin removal beads (My Rem) or kept on ice (H), loaded on a gel and subjected to western blotting using Y188, C1/6.1 and MBP antibodies as well as a total protein stain. The loaded My Rem sample corresponded to 25 times more of the input than the loaded H sample. The 20 kDa band and the 15 kDa MBP band was depleted proportionally more than the FL-APP and APP-CTFα/β bands. (D) Immunodepletion of a human AD brain using an MBP antibody was performed using 2.5 μg (IP 1) or 5 μg (IP 2) MBP antibody or a rabbit IgG (rIgG) as negative control. Five percentage of input before (pre-input) and after (post-input) pre-clearing with beads only, as well of immunodepleted samples were loaded. Whereas the 15 kDa MBP band was efficiently and specifically depleted, the 20 kDa band detected by C1/6.1 was not depleted more by the MBP antibody than by the rIgG.

Figure 6

Detection of the 20 kDa band by other antibodies. An AD human brain homogenate was loaded on a 4%–12% gel and subjected to western blotting using antibodies directed to (A) Keap1 and (B) GC. These antibodies detect bands of their expected molecular weight of 70 and 46–58 kDa, respectively, but also detect a band of approximately 20 kDa.