Proteins were extracted, sonicated, centrifuged at 13,000 rpm for 45 min at 4oC, and supernatants used for immunoblot analysis, as previously described (5 (link),6 ). Actin was always used as an internal loading control. Primary antibodies used were as follows: anti GSAP full length (1:200) and GSAP-16kDa (1:150) (Thermo Scientific, Waltham, MA); anti-TMP21 (1:200), anti-CD-147 (1:200), anti-caspase-3, anti-caspase-7 (1:200) (Santa Cruz Biotech., Dallas, TX), anti-APP (1:200), anti CTF-α and CTF-β (1:200) (Millipore, Billerica, MA, USA); anti-β-actin (1:200). IRDye infrared secondary antibodies were from LI-COR Bioscience.
Anti app
Manufactured by Merck Group
Sourced in United States
Anti-APP is a laboratory equipment product designed to detect the presence of amyloid precursor protein (APP) in biological samples. It functions as a tool for researchers studying Alzheimer's disease and other neurodegenerative conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti bace1, by Merck Group (5 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Irdye infrared secondary antibody, by LI COR (2 mentions)
Anti gsap full length, by Thermo Fisher Scientific (2 mentions)
Gsap 16kda, by Thermo Fisher Scientific (2 mentions)
Anti tmp21, by Santa Cruz Biotechnology (2 mentions)
Anti nicastrin, by Cell Signaling Technology (2 mentions)
Anti sappβ, by Immuno-Biological Laboratories (2 mentions)
Anti actin, by Merck Group (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti cd147, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 7, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 3, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti hemagglutinin ha, by Merck Group (1 mentions)
Anti usp25, by Abcam (1 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
19 protocols using anti app
1
Immunoblot Analysis of Protein Extracts
2
Immunoblot Analysis of Protein Targets
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Immunoblot analysis was performed as previously described (44 (link)). Antibodies were used as follows: anti-USP25 (Abcam, Cambridge, MA, USA; ab187156, 1:1000), anti-APP (Millipore, Billerica, MA, USA; MAB348, 1:1000), anti-Iba1 (Wako Pure Chemical, Osaka, Japan; 016-20001, 1:500), anti-GFAP (glial fibrillary acidic protein) (Cell Signaling Technology, Danvers, MA, USA; 3670, 1:1000), anti–β-III-tubulin (Abcam, ab18207, 1:1000), anti-HA (hemagglutinin) (Sigma-Aldrich, St. Louis, MO, USA; H6908, 1:1000), anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-8017, 1:500), anti–β-actin (Xmbcss, Xiamen, China; bc001, 1:2000), and horseradish peroxidase (HRP)–conjugated secondary antibodies (Thermo Fisher Scientific, 31430 or 31460; 1:3000).
3
Immunoblot Analysis of Alzheimer's Proteins
4
Immunohistochemistry Antibody Panel
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The following antibodies were used. Anti-Rer1, rabbit polyclonal SA5457 and SPY00813 (link); anti-calbindin-D-28K, mouse monoclonal (C-9848), anti-MAP2 mouse monoclonal M4403 and anti-GFAP, rabbit polyclonal (G-9269), all from Sigma; anti-Nav1.6 (ASC-009), anti-Nav1.1 (ASC-001), anti-Cav2.1 (ACC-001), anti-Kv1.1 (APC-009), anti-Kv3.3 (APC-102) and anti-Kv7.2 (APC-050), all rabbit polyclonal from Alomone Labs; anti-ankyrin G mouse monoclonal (sc-12719), Santa Cruz Biotechnology; anti-Scn1b, rabbit polyclonal (AP53815 PU-N), Acris; anti-APP, mouse monoclonal (MAB348), Millipore; anti-PSD-95, mouse monoclonal (6G6-1C9, VAM-PS002), Assay Designs; anti-KCC2, rabbit polyclonal (07–432) and anti-synaptophysin mouse monoclonal (MAB368) both from Millipore, anti-GM130, mouse monoclonal (610822) BD Transduction Laboratories; anti-Sstr3, rabbit polyclonal antibody kindly obtained from Stefan Schulz, Jena45 (link); Alexa Fluor-labeled secondary antibodies, Molecular Probes, HRP-conjugated secondary antibodies, Promega.
5
Immunofluorescent Localization of APP and HA
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Cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 20 min and permeabilized with 0.3% Triton X-100 in PBS. Unspecific binding was inhibited by incubation with blocking solution (1x PBS, 10% BSA and 0.1% Triton X-100) for 15 min. Incubation with primary and secondary antibodies was done for 2 h in PBS containing 1% BSA and 0,1% Triton X-100. Cells were fixed on a glass slide with Immumount (Thermo Scientific, Waltham, MA, USA) and stored at 4°C until analysis. All steps were performed at room temperature. Primary antibodies used were anti-APP (rabbit, 1 : 1000, Upstate, Millipore Corporation, USA) and anti-HA (mouse, 1 : 1000, Covance, New Jersey, USA). Secondary antibodies used were Cy3 coupled anti-rabbit IgG (1 : 1000, Dianova, Hamburg, Germany) and Alexa Fluor 350 coupled anti-mouse IgG (1 : 1000, Moleculare Probes, Life Technologies, Carlsbad, California, USA). Colocalization analysis was performed by MetaMorph software (Molecular Devices, Downingtown, PA).
6
Protective Effects of SalB on AD
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SalB (purity > 99%) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), neurobasal medium, and F12 supplement were obtained from Gibco (New York, NY, USA). 2′,7′-dichlo-rofluorescin diacetate (DCFH-DA) was obtained from Invitrogen (Carlsbad, CA, USA). Assay kits for malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione reductase (GSH-Px) were purchased from Nanjing JianCheng Bioengineering Institute (China). The blots were probed with the following antibodies: anti-APP (Millipore, Boston, MA, USA); anti-sAPPα (Abcam, Cambridge, UK); anti-sAPPβ (Immuno-Biological Laboratories, Fujioka, Japan); anti-BACE1 (Millipore); anti-disintegrin and metalloprotease 10 (ADAM10, Millipore); anti-presenlin 1 (PS1, Millipore), anti-GSK3β (Abcam); anti-pS9-GSK3β (Abcam); anti-β-actin (Sigma-Aldrich, St. Louis, MO, USA); and secondary antibody horseradish peroxidase- (HRP-) conjugated goatanti-rabbit IgG (Cell Signaling Technology, Boston, MA, USA). The Western blot chemiluminescent horseradish peroxidase substrate was purchased from Millipore. All other reagents and chemicals used in the study were of analytical grade.
7
Quantitative Western Blot Analysis of Alzheimer's Biomarkers
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SK-N-BE and HEK-293 cells were homogenized in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.4, 1% Triton X-100, protease inhibitor mixture). Samples were centrifuged at 12,000 g for 15 min at 4 °C and supernatants were collected.
Western blot assay was performed under standard conditions. In brief, equal amounts of proteins were analyzed by 12% SDS-PAGE at 100 V for 90 min and blotted onto nitrocellulose membrane Hybond-enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) in a Bio-Rad Trans-blot apparatus at 350 mA for 3 hours. The blots were probed with anti-p35 (C-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-BACE1 (D10E5, Cell Signaling, Beverly, MA), anti-CDK5 antibody (C-8, Santa Cruz Biotechnology), anti-pAPP (Thr668) (Cell Signaling, Danvers, CA, USA), anti-APP (Millipore, Billerica, MA, USA) and anti GAPDH (Novus biologicals, Littleton, CO, USA) antibodies followed by an alkaline phosphatase-conjugated IgG secondary antibody. Blots were processed by an ECL Plus detection kit as instructed by the supplier (Amersham Biosciences). All experiments were performed at least three times, and the quantization was made using the ImageJ program (National Institute of Health, Bethesda, MD, USA).
Western blot assay was performed under standard conditions. In brief, equal amounts of proteins were analyzed by 12% SDS-PAGE at 100 V for 90 min and blotted onto nitrocellulose membrane Hybond-enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) in a Bio-Rad Trans-blot apparatus at 350 mA for 3 hours. The blots were probed with anti-p35 (C-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-BACE1 (D10E5, Cell Signaling, Beverly, MA), anti-CDK5 antibody (C-8, Santa Cruz Biotechnology), anti-pAPP (Thr668) (Cell Signaling, Danvers, CA, USA), anti-APP (Millipore, Billerica, MA, USA) and anti GAPDH (Novus biologicals, Littleton, CO, USA) antibodies followed by an alkaline phosphatase-conjugated IgG secondary antibody. Blots were processed by an ECL Plus detection kit as instructed by the supplier (Amersham Biosciences). All experiments were performed at least three times, and the quantization was made using the ImageJ program (National Institute of Health, Bethesda, MD, USA).
8
Quantitative Western Blot Analysis
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Cells were lysed in LDS sample buffer (Invitrogen, Germany) including 100 mM dithiothreitol (Roth, Germany) and protease inhibitor mix (Roche, Germany). 20 µg proteins of whole cell lysate were separated on 10% SDS-acrylamide gels and transferred to a nitrocellulose membrane. Blots were either blocked with 5% BSA or milk powder and incubated with primary antibodies diluted in respective blocking buffer as follows: anti-APP (previously described: [35] (link)), anti-ADAM10 (Merck, Germany), anti-GSK3-beta (Bioss, Germany), anti-Pgp (Santa Cruz, Germany) anti-actin (Sigma, Germany), anti-P-ERK and anti-GAPDH (both: Cell Signaling, USA). Detection of APPs-alpha was performed as a dot blot with direct application of cell culture supernatant to the nitrocellulose membrane and 6E10 (Covance, Germany) as primary antibody. Blots were incubated with respective HRP-labeled secondary anti-mouse or anti-rabbit antibodies (Thermo Scientific, Germany) and GAPDH or actin were used as loading controls. Signals were detected with a CCD-camera imaging system and quantitatively analyzed with AIDA image analyzer 4.26 software (Raytest, Germany).
9
Comprehensive Antibody Validation for Alzheimer's Research
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Primary antibodies included anti-Aβ 4G8 (SIG-39220, BioLegend, San Diego, CA, USA), anti-SPIN90 (generated in our laboratory), anti-Rab11 (ab3612, Abcam, Cambridge, UK), anti-GFP (sc-9996, Santa Cruz Biotechnology, Dallas, TX, USA), anti-APP (MAB348, EMD Millipore, Darmstadt, Germany), anti-BACE1 (B0681, Sigma, St. Louis, MO, USA), anti-Nicastrin (5665, Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p35/p25 (2680, Cell Signaling Technology, MA, USA) and anti-tubulin (T6199, Sigma, St. Louis, MO, USA). Secondary antibodies included horseradish peroxidase (HRP)-conjugated donkey anti-mouse (115-035-006) and anti-rabbit antibodies (111-035-006, Jackson Laboratory, Bar Harbor, ME, USA). Unless otherwise noted, all chemicals were purchased from Sigma.
10
Proteomic analysis of Alzheimer's markers
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The following primary antibodies were used in this study: anti-Aβ (82E1, IBL, Gunma, Japan; 6E10,Covance, Emeryville, CA, USA), anti-BACE1 (AB5832, Merck Millipore, Darmstadt, Germany; D10E5, Cell Signaling, Danvers, MA, USA), anti-APP (R37 [50 (link)]; 22C11, Merck Millipore), anti-ADAM10 (Sigma, St Louis, MO, USA), anti-phospho-eIF2α (Ser51) (Cell Signaling), anti-eIF2α (Assay Biotechnology, Sunnyvale, CA, USA), anti-cleaved caspase 3 (Asp175) (Cell Signaling), anti-GRP78 (BD Biosciences, San Jose, California, USA), anti-β-actin (Sigma), anti-MAP2 (Merck Millipore), and antibody to the rhodopsin tag (1D4) [51 (link)] obtained from University of British Columbia.
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