Bace1

Manufactured by Abcam

Sourced in United States, United Kingdom

BACE1 is an enzyme that plays a key role in the production of amyloid-beta peptides, which are the main components of amyloid plaques found in the brains of individuals with Alzheimer's disease. This product is a laboratory tool that can be used to study the activity and regulation of BACE1 in various experimental settings.

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Lab products found in correlation

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Close spelling variants of this product

Anti-BACE1 (12 citations) Anti-BACE1 antibody (4 citations) Rabbit-anti-BACE1 (3 citations) Monoclonal anti-BACE1 (ab108394) (2 citations) BACE1) antibody (2 citations) BACE1 (61-3E7, (2 citations)

20 protocols using bace1

Western Blot Analysis of Brain Tissue

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Brain tissue lysates for WB were incubated on ice for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. The protein concentration in the supernatant was quantified using a BCA assay kit (23,227, Thermo Fisher Scientific). Equal amounts of protein (20 μg) in the supernatant were separated by 8% SDS-PAGE gel, and proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) after electrophoresis. The membrane was blocked with 5% nonfat dried milk at RT for 1.5 h, followed by incubation with primary antibodies against sAPPβ, sAppα (IBL, Fujioka, Japan), BACE-1 (Abcam, Cambridge, MA), GFAP (Millipore, Burlington, MA, USA), and β-actin (Boster, Wuhan, China) as control proteins at 4°C overnight. After three washes with 1× TBST, the membrane was incubated in horseradish peroxidase-conjugated secondary antibody (ZSGB-BIO, Beijing, China) at RT for 1 h. Membranes were incubated in Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA), and visualized with the Gel Doc XR System (Bio-Rad) and analyzed using AlphaEaseFC software.

Liang Z., Li X., Luo X., Luo H., Chen Y., Cai M., Zhong X., Fang Y., Guo T., Shi Y, & Zhang X. (2022). The Aptamer Ob2, a novel AChE inhibitor, restores cognitive deficits and alleviates amyloidogenesis in 5×FAD transgenic mice. Molecular Therapy. Nucleic Acids, 28, 114-123.

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Western Blot Analysis of Alzheimer's Markers

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Cells were analyzed by western blotting using 4–12% SDS gradient gels (NP0322PK2; Invitrogen) and the following antibodies were used: APP (Abcam, ab32136, Cambridge, UK), BACE1 (Abcam, ab183612), PSEN1 (Cell Signaling Technology, D39D1, Danvers, MA, USA), cleaved Notch1 (Cell Signaling Technology, 4147), Aβ1-42 (Abcam, ab201060), and anti-β-actin (Abcam, ab8227). Electrophoreses were performed using 15% Tris-Glycine SDS gels for Aβ and 12% Tris-Glycine SDS gradient gels for PSEN1 in 12-well gel, and the resulting bands were transferred by western blotting. The protein concentration of the loading sample was 30 μg. Immunoreactivity was analyzed by chemiluminescence (GE Healthcare, Piscataway, NJ, USA). The protein marker ranges from 10–180 kDa were used (Tris-glycine-SDS running buffer) (GeneDireX, PM006-0500, Taoyuan City, Taiwan). The transfer buffer was 25% Methanol and 10% TG-SDS buffer (Avantor, 0783-5L, Radnor, PA, USA). The setup time and voltage are 90 min and 400 mA by gel transfer to PVDF member. The chemiluminescence signal was observed using a digital image analyzer (Fuji Film Inc, LAS-3000, Tokyo, Japan).

Wuli W., Lin S.Z., Chen S.P., Tannous B.A., Huang W.S., Woon P.Y., Wu Y.C., Yang H.H., Chen Y.C., Fleming R.L., Rogers J.T., Cahill C.M., Ho T.J., Chiou T.W, & Harn H.J. (2022). Targeting PSEN1 by lnc-CYP3A43-2/miR-29b-2-5p to Reduce β Amyloid Plaque Formation and Improve Cognition Function. International Journal of Molecular Sciences, 23(18), 10554.

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Quantifying APP, APP Fragments, and BACE1 in Cortical Homogenates

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Cortical RAB high-salt homogenates that had been retained prior to centrifugation as described above were utilized for immunoblot analyses of APP holoprotein, BACE1 and APP carboxy-terminal C99/C89 and C83 fragments. 0.1 ml of cortical homogenates were treated with 25 μl of 10% SDS to solubilize membrane proteins, followed by centrifugation at 100,000 × g for 20 min at 4⁰C. The supernatant was collected, and the pellet was re-extracted with 0.1 ml of 10% SDS in water. After centrifugation, this supernatant was combined with the first supernatant sample, and the total protein determined by BCA. A total of 30 μg of protein from each study mouse underwent SDS-PAGE (4–12% gradient gels), followed by blotting to nitrocellulose membranes as previously described [50 (link)]. Intact APP holoprotein and carboxy-terminal APP fragments were detected using the carboxy-terminal APP monoclonal antibody 5685 [48 (link)], as previously described [50 (link)]. BACE1 was also detected (Abcam), and GAPDH (Advanced Immunochemical) was used as a loading control. Secondary antibodies and imaging were essentially as described [50 (link)], using the Odyssey Imaging System (LI-COR Biosciences, Lincoln, NE). All APP, APP fragments and BACE1 integrated signals were normalized to GAPDH integrated signal for each sample. The quantification was conducted by an individual masked to the blot lane assignments.

Yao Y., Nzou G., Alle T., Tsering W., Maimaiti S., Trojanowski J.Q., Lee V.M., Ballatore C, & Brunden K.R. (2020). Correction of microtubule defects within Aβ plaque-associated dystrophic axons results in lowered Aβ release and plaque deposition. Alzheimer's & dementia : the journal of the Alzheimer's Association, 16(10), 1345-1357.

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Alzheimer's Disease Biomarkers Analysis

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Monoclonal antibodies IR, PI3K, p-NF-κB^Ser536, p-GSK3β^Ser9, GSK3β, GLUT3, PKC𝜀, PKCη, BACE-1, APP, glial fibrillary acidic protein (GFAP) and polyclonal antibody Aβ42 were purchased from Abcam. Monoclonal antibodies p-IRS-1^Ser307, p-AKT^Ser473, AKT, p-JNK^{Thr183/Tyr185}, JNK, p-4E-BP1^Thr37/46, and NF-κB were obtained from CST. P-PI3K P85^Tyr467 was purchased from absin. P-IKKβ^Y199 was purchased from Gene Tex. P-ERK1/2 ^{Thr202/Tyr204} and ERK1/2 were purchased from Millipore. The ladder marker was obtained from Thermo scientific. STZ was purchased from biosci biotechnology Co.; Ltd. ELISA kits were purchased from BOSTER.

Chen Q., Mo R., Wu N., Zou X., Shi C., Gong J., Li J., Fang K., Wang D., Yang D., Wang K, & Chen J. (2017). Berberine Ameliorates Diabetes-Associated Cognitive Decline through Modulation of Aberrant Inflammation Response and Insulin Signaling Pathway in DM Rats. Frontiers in Pharmacology, 8, 334.

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Western Blot Analysis of Alzheimer's Proteins

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Brain tissues were homogenized and sonicated in ice‐cold lysis buffer [50 mM Tris–HCl, pH 8.0, 140 mM NaCl; 1.5 mM MgCl₂; 0.5% NP‐40 with complete protease inhibitor cocktail (Roche)]. Protein concentration was measured in the supernatant by BCA Protein Assay (Dingguo). The monoclonal or polyclonal antibodies used were BACE1 (Abcam, 1:1000); APP full length (A8717, Sigma, 1:3000); sAPPα (6E10, Covance, 1:1000); sAPPβ (Covance, 1:500); ADAM10 (Abcam, 1:1000); PSEN1 (Proteintech, 1:500); GAPDH (Proteintech, 1:10000); tau46 (CST, 1:1000); phosphorylation‐tau 181 (CST, 1:1000); phosphorylation‐tau 202 (Abcam, 1:5000); and phosphorylation‐tau 396 (Abcam, 1:10000). The secondary antibodies were goat anti‐rabbit or anti‐mouse horseradish peroxidase‐labeled antibodies (Proteintech, 1:5000). The membranes were visualized using an ECL reagent (Thermo) and a Fusion FX5 image analysis system (VilberLourmat). Relative protein intensities were calculated using Quantity One software (Bio‐Rad).

Luo B., Chen J., Zhou G., Xie X., Tang J., Wen Q., Song L., Xie S., Long Y., Chen G, & Hu X. (2023). Apicidin attenuates memory deficits by reducing the Aβ load in APP/PS1 mice. CNS Neuroscience & Therapeutics, 29(5), 1300-1311.

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Western Blot Analysis of Neuroinflammation

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The brain tissues, treated astrocytes, and microglial BV-2 cells were prepared as previously described (Gu et al. 2015 (link)). An equal amount of total protein (20 µg) was resolved on 8–15% sodium dodecyl sulfate polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P; pore size 0.45 µm, EMD Millipore, USA). The membranes were blocked for 1 h in 5% skim milk solution and incubated for overnight at 4 °C with specific antibodies. To detect target proteins, specific antibodies against APP, iNOS (1:1000, Novus Biologicals, Inc., Littleton), BACE1, IBA-1 (1:1000, Abcam, Inc., Cambridge, MA, USA), COX-2 (1:1000, Cell Signaling Technology, Inc., Beverly, MA, USA), GFAP, p50, p65, IκB, phospho-IκB, β-actin, Histone H1 (1:1000, Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA) were used. The blots were then incubated with one of the following corresponding conjugated secondary antibodies: goat anti-rabbit, goat anti-mouse, or donkey anti-goat IgG-HRP (1:5000; Santa Cruz Biotechnology Inc. Santa Cruz, CA, USA). Immunoreactive proteins were detected with an enhanced chemiluminescence Western blotting detection system. The relative density of the protein bands was scanned by densitometry using MyImage (SLB, Seoul, Korea) and quantified by Labworks 4.0 software (UVP Inc., Upland, CA, USA).

Gu S.M., Lee H.P., Ham Y.W., Son D.J., Kim H.Y., Oh K.W., Han S.B., Yun J, & Hong J.T. (2018). Piperlongumine Improves Lipopolysaccharide-Induced Amyloidogenesis by Suppressing NF-KappaB Pathway. Neuromolecular Medicine, 20(3), 312-327.

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Whole Cell Lysate Preparation and Western Blot

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Whole cell lysate was prepared by adding 5μL/mg tissue of ice cold lysis buffer (20mM HEPES, 1% Triton x-100, 1% sodium deoxycolate, 1mM DTT, 1% SDS, 1X protease inhibitors (Thermo Scientific Pierce Protein Biology), 1 mM NaF, 5 mM EDTA, 10 mM β-glycerophosphate and 250 μM Na₃VO₄) and sonicated 3 times for 5 seconds on ice, clarified by centrifugation at 15,000 × g for 12 minutes at 4°C and stored at −80°C until use. Protein concentration was measured using detergent compatible protein quantification kit (Biorad) and final loading samples were prepared at 4μg/μL protein in LDS Sample Buffer (Life Technologies) with 50 mM DTT (Life Technologies). Sixty μg of whole cell lysate were loaded into each well of a NuPAGE NOVEX 4–12% Bis-Tris gel with MES running buffer, transferred onto Immobilon-FL PVDF (Millipore). A solution of 5% milk in TBS + 0.1% Tween 20 was used to block and dilute primary and secondary antibodies (1:5000, LiCor Biosciences, Rabbit antibody conjugated with IRdye 800, mouse antibody conjugated with IRdye 680), scans and quantifications were used using a LiCor Odyssey scanner. The primary antibodies included: ADAM10 (Rb 1:1000, Abcam ab1997), GFAP (Rb 1:50,000, Abcam ab7260), Actin (Ms 1:2000, Abcam ab3280), BACE1 (Rb 1:1000, Abcam ab2077).

Pawlosky R.J., Kemper M.F., Kashiwaya Y., King M.T., Mattson M.P, & Veech R.L. (2017). Effects of a dietary ketone ester on hippocampal glycolytic and TCA cycle intermediates and amino acids in a 3xTgAD mouse model of Alzheimer’s disease. Journal of neurochemistry, 141(2), 195-207.

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Berberine Biomarker Analysis Protocol

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Monoclone antibodies IR, PI3K P85, p-NF-κB p65, IKK, BACE-1, APP, α7nAChR and polyclone antibody Aβ were purchased from Abcam (Cambridge, MA, USA). Monoclone antibody p-Akt (Ser473), AKT, NF-κB, p-IKK, p-IRS-1(Ser307), and IRS-1 were purchased from Cell Signaling Technology (Boston, MA, USA). Insulin ELISA kit (EZRMI-13) and PVDF membrane (0.45 µm) were obtained from Millipore (Billerica, MA, USA). The cytokines of IL-1β, IL-18 and TNF-α were purchased from BOSTER (Wuhan, China) and the ACh kits (A105-1: tissue, A105-2: Serum) and the AChE kits (A024) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The ladder marker was obtained from Thermo Scientific (Waltham, MA, USA). Finally, the GLU kit was purchased from Shanghai Mind Bioengineering Co., Ltd. (Shanghai, China). Berberine was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (99% pure, Shanghai, China). All other reagents purchased from located market were of analytical grade.

Wang K., Chen Q., Wu N., Li Y., Zhang R., Wang J., Gong D., Zou X., Liu C, & Chen J. (2019). Berberine Ameliorates Spatial Learning Memory Impairment and Modulates Cholinergic Anti-Inflammatory Pathway in Diabetic Rats. Frontiers in Pharmacology, 10, 1003.

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Western Blotting of Neural Proteins

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Western blotting was completed as previously described [27 (link)]. Whole cell lysates were generated using RIPA buffer with protease and phosphatase inhibitors (Sigma and ThermoFisher). Briefly, equal amount of protein were resolved via SDS-PAGE on Criterion TGX gels 4–15% (BioRad). Gels were transfered to PVDF membranes and blocked with 5% BSA in PBST. Primary antibodies were incubated overnight at 4°C followed by three washes with PBST. Secondary antibodies (ThermoFisher) were incubated at room temperature for one hour. Gels were imaged using WestFemto ECL (ThermoFisher) and a BioRad ChemiDoc XRS imaging system. Loading controls included either actin (Cell Signaling) or AmidoBlack (Sigma) total protein stain. Primary antibodies include AβPP 6E10 (Biolegend), BACE1 (Abcam), COX41I (Cell Signaling), Calreticulin (Cell Signaling), GAPDH (Cell Signaling), GFAP (Abcam), HDAC (Cell Signaling), MAP2 (Abcam), Nestin (ThermoFisher), S100β (Abcam), and Synaptophysin (Abcam).

Wilkins H.M., Troutwine B.R., Menta B.W., Manley S.J., Strope T.A., Lysaker C.R, & Swerdlow R.H. (2022). Mitochondrial Membrane Potential Influences Amyloid Precursor Protein Localization and Aβ Secretion. Journal of Alzheimer's disease : JAD, 85(1), 381-394.

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Immunofluorescence Analysis of ARPE-19 Cells

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Human RPE cell line ARPE-19 cells were fixed with 4% paraformaldehyde in PBS for 15 min, permeated in 0.05% Triton-X 100 for 15 min and blocked with 4% FBS in PBS for 30 min. Anti-ZO-1 antibody (Abcam, Cambridge, UK) was used to determine the expression of junctional proteins. Anti-Amyloid β 42 (Aβ42) (Abcam) and anti-BACE1 against β-secretase (BACE1, Abcam) antibodies were used to determine the expression of amyloid β peptides. Hoechst 33,342 or DAPI (40,6-diamidino-2-phenylindole, Thermo Fisher Scientific) was used to stain nucleic acids for the nuclear staining. Images on slides were taken using a fluorescence microscope system.

Chen S.J., Lin T.B., Peng H.Y., Liu H.J., Lee A.S., Lin C.H, & Tseng K.W. (2021). Cytoprotective Potential of Fucoxanthin in Oxidative Stress-Induced Age-Related Macular Degeneration and Retinal Pigment Epithelial Cell Senescence In Vivo and In Vitro. Marine Drugs, 19(2), 114.

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