- AHB0272 - Invitrogen Antibodies APP antibody

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

Haytural H, Lundgren JL, Köse TB, Jordà-Siquier T, Kalcheva M, Seed Ahmed M, Winblad B, Sundström E, Barthet G, Tjernberg LO, Frykman S
Frontiers in aging neuroscience 2019;11:273

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Beta Amyloid was performed using membrane enriched extract (30 µg lysate) of PC-3 (Lane 1) and tissue extract (30 µg lysate) of Rat Brain (Lane 2). The blots were probed with Anti-Beta Amyloid Mouse Monoclonal Antibody (Product # AHB0272, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 125 kDa isoform corresponding to Beta Amyloid was observed in PC-3 and, another isoform of 34 kDa was observed in Rat Brain. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Beta Amyloid was performed using membrane enriched extract (30 µg lysate) of PC-3 (Lane 1) and tissue extract (30 µg lysate) of Rat Brain (Lane 2). The blots were probed with Anti-Beta Amyloid Mouse Monoclonal Antibody (Product # AHB0272, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 125 kDa isoform corresponding to Beta Amyloid was observed in PC-3 and, another isoform of 34 kDa was observed in Rat Brain. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of beta-Amyloid was performed using U-87 MG cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with beta-Amyloid Mouse Monoclonal Antibody (AHB0272, red histogram) or with mouse isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 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)-beta (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.

AHB0272

Antibody data