Ab108394

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab108394 is a monoclonal antibody that recognizes a specific antigen. It is intended for use in research applications.

Automatically generated - may contain errors

Lab products found in correlation

Nitrocellulose membrane, by Cytiva (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Mn1020b, by Thermo Fisher Scientific (1 mentions) G3893, by Merck Group (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Ab32136, by Abcam (1 mentions) β actin 66009 1 ig, by Proteintech (1 mentions) Prolong gold, by Thermo Fisher Scientific (1 mentions) A1r confocal microscope, by Nikon (1 mentions) Ab19862, by Abcam (1 mentions) Mn1020, by Thermo Fisher Scientific (1 mentions) Ab201060, by Abcam (1 mentions)

6 protocols using ab108394

Immunohistochemistry and Immunofluorescence of Alzheimer's Pathologies

Cited 2 times

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IHC and IF were performed as previously described^{12 (link),14 (link)}. For IHC staining of NP-tau and Aβ, primary antibodies AT8 (Thermo Fisher Scientific, MN1020B, 1:500) and HJ3.4 (2 μg/ml in-house) respectively were used.
For IF staining, co-stains were performed for X34, BACE1, and AT8. Fibrillar Aβ was stained with X34 dye (SML-1954) and antibodies to AT8 and BACE1 (abcam, ab108394, 1:500) were used to evaluate peri-plaque pathologies.

Gratuze M., Jiang H., Wang C., Xiong M., Bao X, & Holtzman D.M. (2022). APOE immunotherapy mitigates cerebral Aβ accumulation and Aβ-associated tau seeding and spreading in a mouse model of amyloidosis. Annals of neurology, 91(6), 847-852.

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Astrocytes and BACE1 Interaction Assay

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To confirm the relationship between both GFAP⁺ astrocytes and BACE1, we immunoprecipitated BACE1 and then the co-immunoprecipitated GFAP. Briefly, samples were lysed in 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% NP40, 1 nM orthovanadate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride with a protease inhibitor cocktail (Sigma-Aldrich). The lysates were clarified by centrifugation at 14,000 rpm for 5 min. A Pierce Protein Quantification Assay was performed, and then 200 μg total protein was incubated overnight at 4°C in the presence of the anti-BACE1 antibody (ab108394, Abcam, 1:100). Protein G Sepharose beads were added, and the samples were incubated for an additional 2 h at room temperature. The immune complexes were washed three times using immunoprecipitation lysis buffer before SDS-PAGE and immunoblotting. The proteins were separated using 10% SDS-PAGE, transferred to nitrocellulose membranes (Amersham) and probed with mouse anti-GFAP antibody (G3893, Sigma-Aldrich, 1:250). Whole lysates were used as positive controls, and incubation with an IgG peptide (395040065, Thermo, 1:500) was used as a negative control for immunoprecipitation. The blots were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences). To minimize interassay variation, samples from all experimental groups were run in parallel.

Chacón-Quintero M.V., Pineda-López L.G., Villegas-Lanau C.A., Posada-Duque R, & Cardona-Gómez G.P. (2021). Beta-Secretase 1 Underlies Reactive Astrocytes and Endothelial Disruption in Neurodegeneration. Frontiers in Cellular Neuroscience, 15, 656832.

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Alzheimer's Disease Protein Detection Protocol

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Maintain diet and 2% of HMD were obtained from Synergetic Pharmaceutical Bioengineedring Co., Lt. (China). HMD was prepared by adding 2% Met to the Maintain diet. Amyloid precursor protein (APP, ab32136), beta-secretase 1 (BACE1, ab108394), insulin-degrading enzyme (IDE, ab109538), amyloid-β 1-42 (Aβ_1-42, ab201061), DNA methyltransferase1 (DNMT1, ab188453), and DNA methyltransferase 3a (DNMT3a, ab188470) antibodies were purchased from Abcam (UK). Amyloid-β 1-40 (Aβ_1-40, D8Q7I) and 5-mC (D3S2Z) antibodies were obtained from Cell Signaling Technology (USA). Neprilysin (NEP, D161012) antibody was obtained from Sangong Biotech (China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 10494-1-AP) and β-actin (66009-1-Ig) antibodies were obtained from Proteintech (China). HRP-conjugated affinipure goat anti-rabbit IgG (SA00001-2), HRP-conjugated affinipure goat anti-mouse IgG (SA00001-1), and CoraLite488- conjugated affinipure goat anti-rabbit IgG (SA00013-2) were obtained from Proteintech (China).

Pi T., Wei S., Jiang Y, & Shi J.S. (2021). High Methionine Diet-Induced Alzheimer's Disease like Symptoms Are Accompanied by 5-Methylcytosine Elevated Levels in the Brain. Behavioural Neurology, 2021, 6683318.

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Immunostaining of Amyloid-Beta and BACE1

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Paraformaldehyde-fixed brains were sectioned at 30 μm. Free-floating sagittal (females) or coronal (males) sections were treated with TBS-0.25% Triton X-100 with 16 mM glycine for 60 minutes on a shaker. Sections were washed in TBS, blocked in 5% donkey serum in TBS-0.25% Triton X-100 for one hour, then washed 2x10 minutes in 1% BSA in TBS-0.25% Triton X-100. Sections were incubated at 4°C on a shaker overnight with anti-Aβ 3D6 (mouse monoclonal, 1:250, gift from Lisa McConlogue, Elan). The following morning, anti-BACE1 (1:250, rabbit monoclonal, Abcam ab108394) was added and the sections were incubated an additional 2 hours at 37°C with shaking. The sections were washed 3 times with TBS then incubated for 1.5 hours at room temperature on a shaker with Invitrogen Alexa Fluor secondary antibodies at 1:250 (Donkey anti-Rabbit 594 and donkey anti-mouse 488) and DAPI at 300 nM. The sections were then mounted on slides and immediately cover-slipped using ProLong Gold (Invitrogen). Confocal images were captured on a Nikon (Tokyo, Japan) A1R confocal microscope with a 40x objective, and 12 images were stitched together using NIS Elements software to generate a larger field image. All images were captured at same laser and software configurations.

Sadleir K.R., Eimer W.A., Cole S.L, & Vassar R. (2015). Aβ reduction in BACE1 heterozygous null 5XFAD mice is associated with transgenic APP level. Molecular Neurodegeneration, 10, 1.

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Hippocampal Protein Extraction and Immunoprecipitation

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The hippocampus samples were lysed in 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1% NP40, 1 nM orthovanadate, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aldrich; Cardona-Gomez et al., 2004 (link)). The lysates were clarified by centrifugation at 14,000 rpm for 5 min. A protein assay was performed, and 200 μg of protein was incubated overnight at 4°C in the presence of BACE1 Ab (ab108394, Abcam, 1:100). Protein G-sepharose beads were added, and the samples were incubated for an additional 2 h at RT. The immune complexes were washed three times using immunoprecipitation (IP) lysis buffer before SDS-PAGE and immunoblotting. The proteins were separated using 10% SDS-PAGE, transferred onto nitrocellulose membranes (Amersham), and probed with anti-human PHF-tau (AT8, MN1020, Thermo, 1:250), rabbit cPLA2 (ab198898, Abcam, 1:250), and mouse SCD1 (ab19862, Abcam, 1:250). The lysates were used as the positive control, and incubation with the IgG peptide was used as the negative control for IP.

Bedoya-Guzmán F.A., Pacheco-Herrero M., Salomon-Cruz I.D., Barrera-Sandoval A.M., Gutierrez Vargas J.A., Villamil-Ortiz J.G., Villegas Lanau C.A., Arias-Londoño J.D., Area-Gomez E, & Cardona Gomez G.P. (2023). BACE1 and SCD1 are associated with neurodegeneration. Frontiers in Aging Neuroscience, 15, 1194203.

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Analysis of Alzheimer's-related proteins

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Exosomes or sEVs were lysed in the isopyknic lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, and 1 mM Phenylmethanesulfonyl fluoride (PMSF) on ice for 10 min. The samples were then boiled for 10 min after adding loading buffer, and the protein concentrations were determined by BCA method. Equal amounts (10 μg) of proteins underwent SDS-PAGE and were electroblotted onto PVDF membranes. The membranes were then blocked with 5% nonfat milk in PBS-Tween 20 (PBST; 0.05%) for 1 h and incubated with primary antibody (1:1000 in PBST) at 4 °C overnight. Antibody for APP (#2452S) was obtained from Cell Signaling Technology (CST, Danvers, MA, USA); antibodies for Aβ42 (Ab201060) and BACE1 (Ab108394) were from Abcam (Abcam, Cambridge, MA, USA). After being washed three times in PBST (0.1% Triton X-100 in PBS), the membranes were incubated with appropriate secondary antibodies (1:5000) at room temperature for 1 h. After washing, the immunoreactive bands were visualized by 3,3′-diaminobenzidine (DAB). The integrated densities were analyzed by Image J software (NIH, Bethesda, MD, USA).

Dong Z., Gu H., Guo Q., Liu X., Li F., Liu H., Sun L., Ma H, & Zhao K. (2022). Circulating Small Extracellular Vesicle-Derived miR-342-5p Ameliorates Beta-Amyloid Formation via Targeting Beta-site APP Cleaving Enzyme 1 in Alzheimer’s Disease. Cells, 11(23), 3830.

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