N1660

Manufactured by Merck Group

Sourced in China, United States

The N1660 is a lab equipment product manufactured by Merck Group. It is designed to perform specific laboratory functions, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information about the intended use or capabilities of this product is not available.

Automatically generated - may contain errors

Lab products found in correlation

Ab32136, by Qiagen (2 mentions) Mab348, by Fortrea (2 mentions) Anti pen2, by Merck Group (1 mentions) Anti a2ar, by Merck Group (1 mentions) Anti eea1, by BD (1 mentions) Anti flag, by Merck Group (1 mentions) Anti ha, by Merck Group (1 mentions) Prb 354p, by Fortrea (1 mentions) Prb 550p, by Fortrea (1 mentions) Ab6276, by Abcam (1 mentions) Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions) Dynabeads protein g, by Thermo Fisher Scientific (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions) Pgem 1, by Promega (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) Deoxynucleotide, by Promega (1 mentions) Tnt quick transcription translation system, by Promega (1 mentions) 35s met, by PerkinElmer (1 mentions) Oligonucleotides, by Eurofins (1 mentions) E8032, by New England Biolabs (1 mentions)

7 protocols using n1660

Quantification of Gamma-Secretase Complex

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Anti-PS1-N-terminus (1–65) (PRB-354P, 1:1,000, Covance, Davis, CA, USA); Anti-PS1-N-terminus (MAB1563, 1:100, EMD Millipore, Darmstadt, Germany); anti-PS1 loop (263–407) (529592, 1:2,000, Calbiochem); anti-APH1aL/C terminal (245–265) (PRB-550P, 1:1,000, Covance); anti-NCT (N1660, 1:1,000, Sigma), Anti-Pen2 (P5622, 1:1,000, Sigma); anti-BACE1 (AP7774b, 1:1,000, Abgent, Suzhou, China); anti-HA (H6908, 1:5,000, Sigma); anti-Flag (F3156, 1:2,000, Sigma); anti-A_2AR (05–717, 1:1,1000, Millipore); anti-EEA1 (610457, dilution 1:200, BD transduction laboratories).

Lu J., Cui J., Li X., Wang X., Zhou Y., Yang W., Chen M., Zhao J, & Pei G. (2016). An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity. PLoS ONE, 11(11), e0166415.

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Validating Antibody Specificity for Nicastrin

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We used a commercially available rabbit anti-human IgG polyclonal antibody that targets human nicastrin (#N1660; Sigma-Aldrich, St. Louis, MO, USA; RRID:
AB_477259) which is has performed well in some previous publications
^{13 (link),
14 (link)}. The antibody was raised against Uniprot nicastrin peptide
Q92542 (709 amino acid total size). The polyclonal was generated by challenging rabbits with a synthetic peptide corresponding to the C-terminal cytoplasmic domain of nicastrin (peptides 693-709) fused with keyhole limpet hemocyanin as an adjuvant.
The technical documentation claims this subsequence is identical to the matching region of nicastrin in mouse. However, aligning Q92542 to the primary mouse nicastrin peptide sequence (
NP_067620.3) with Clustal Omega
^{15 (link),
16 (link)} actually shows 1 mismatch (94.1% identity;
Figure 1). It’s unclear if this discrepancy is due to changes to either the human or mouse peptide sequence for the most common isoform over time as the references have been updated.
We used a mouse anti-human beta actin monoclonal antibody (#AB6276; Abcam, Cambridge, MA, USA; RRID:
AB6276&l=AB6276">AB_2223210) as a loading control. The details of all primary and secondary antibodies are summarized in
Table 1.

Mesa R.A, & Roberson E.D. (2020). Validation of a commercial antibody to detect endogenous human nicastrin by immunoblot. F1000Research, 8, 1211.

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BCMA Immunoprecipitation and Western Blot Analysis

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Immunoprecipitation of BCMA was performed with the monoclonal antibody A7D12.2, the monoclonal antibody C12A3.2 or a polyclonal goat antibody (AF193; R&D Systems). These antibodies were coupled to Dynabeads Protein G (Life Technologies, AS, Oslo) and cross-linked with bis-sulfosuccinimidyl-suberate3 (link) (Pierce Chemical Co., Rockford, IL). After successive incubation with either supernatant of plasmacytoma cells or serum, we eluted with glycine or SDS loading buffer (NuPAGE LDS Sample Buffer, Life Technologies). Cells were lysed at 4 °C for 1 h in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris pH 7.5, 1% Nonidet P-40) containing complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). BCMA was detected by western blot analysis with the monoclonal antibody C12A3.2. Blots were developed using the mouse true blot goat anti-mouse IgG-HRP system (EBioscience) and enhanced chemiluminescence (ECL). Expression and endoproteolysis of PS1 was analysed by immunoblotting of cell lysates with the antibody PSEN1 (Epitomics); maturation of nicastrin (NCT) was evaluated by immunoblotting with the antibody N1660 (Sigma-Aldrich).

Laurent S.A., Hoffmann F.S., Kuhn P.H., Cheng Q., Chu Y., Schmidt-Supprian M., Hauck S.M., Schuh E., Krumbholz M., Rübsamen H., Wanngren J., Khademi M., Olsson T., Alexander T., Hiepe F., Pfister H.W., Weber F., Jenne D., Wekerle H., Hohlfeld R., Lichtenthaler S.F, & Meinl E. (2015). γ-secretase directly sheds the survival receptor BCMA from plasma cells. Nature Communications, 6, 7333.

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Immunoblotting and Aβ Quantification

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The following antibodies were used for immunoblotting: NT-1 recognizing the PS1-NTF [61] (link), α-loop (Chemicon) raised against PS1-CTF, N1660 (Sigma) raised against the C-terminal of nicastrin, 3891 (ProSci Inc.) raised against Pen-2 and α-GAPDH (Acris GmbH) recognizing GAPDH. For the quantification of secreted Aβ38, 40 and 42 with Meso Scale Discovery (MSD) technology, C-terminal specific antibodies (Aβ 1-x) were used and detection was performed by SULFO-TAG™ 6E10 antibody. For quantification of sectreted Aβ40 and Aβ43 using Aβ40 Wako II ELISA kit (Wako Chemicals GmbH) and FL 1-43 ELISA kit (Immuno-biological Laboratories), respectively, the capture antibody was BNT77 for Aβ40 and Aβ38-43 for Aβ43. Detection antibodies were BA27 (Aβ40) and 82E1 (Aβ43), respectively. Unless otherwise stated, all chemicals were from Sigma–Aldrich. Plasmid pGEM1, TNT® Quick transcription/translation system, and deoxynucleotides were purchased from Promega and ³⁵S-Met from PerkinElmer. All enzymes were obtained from Fermentas except Phusion DNA polymerase that was from Finnzymes. Oligonucleotides were from Eurofins MWG Operon.

Wanngren J., Lara P., Öjemalm K., Maioli S., Moradi N., Chen L., Tjernberg L.O., Lundkvist J., Nilsson I, & Karlström H. (2014). Changed membrane integration and catalytic site conformation are two mechanisms behind the increased Aβ42/Aβ40 ratio by presenilin 1 familial Alzheimer-linked mutations. FEBS Open Bio, 4, 393-406.

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In vitro Gamma-Secretase Complex Assay

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In vitro IP activity assays were performed by capturing γ-secretase complexes using either an anti-nicastrin antibody (Sigma N1660) bound to protein A Dynabeads or an anti-MBP antibody (New England Biolabs E8032) bound to protein G Dynabeads at 4°C for 1.5 hours, followed by 3 washes with 0.25% CHAPSO, 50 mM HEPES, pH7.2, 150 mM NaCl. The resin was resuspended in 50 μl of phosphatidylcholine (1 mg/ml), 0.25 mg/ml phosphatidylethanolamine, 0.25% CHAPSO (in 50 mM HEPES, pH7.2, 150 mM NaCl), and 1 μM C100-FLAG substrate was added and incubated at 37°C for 3 hr. Bound samples were eluted with Laemmli sample buffer. Input lysate, eluted material and activity assay supernatant were all analyzed by SDS-PAGE Western blots probed with mouse anti-Nicastrin (1:1,000; BD Transduction Laboratories), rabbit anti-Nicastrin (1:2,000 Sigma N166), mouse anti-MBP (1:10,000 NEB E8032), rabbit anti-PS1-NTF (1:5,000 B19; kind gift of B. De Strooper), rabbit anti-PS1-CTF (1:2,000; Abcam 76083), rabbit anti-Pen-2 (1:5,000; Abcam 154830), or mouse anti-FLAG (Sigma F3165). All Western blots were scanned on an Odyssey Infrared Imaging System (Li-Cor), and densitometry analysis using Odyssey software.

Holmes O., Paturi S., Wolfe M.S, & Selkoe D.J. (2014). Functional Analysis and purification of a Pen-2 Fusion Protein for γ-Secretase Structural Studies. Journal of neurochemistry, 131(1), 94-100.

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Alzheimer's Antibody Characterization Protocol

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Antibodies to PS1 N‐terminal (PS1N, Capell et al, 1997, immunoblot (IB) 1:1,000) and PS2 C‐terminal fragments (BI.HF5c, Steiner et al, 1999, IB: 1:2,000), respectively, as well as antibodies to the C‐terminus of APP (6687, Steiner et al, 2000, IB 1:1,000), and to total Aβ (3552, Yamasaki et al, 2006, immunoprecipitation (IP) 1:500–1:7,500 and 2D8, Shirotani et al, 2007, IB 3 μg/ml), have been described previously. Neoepitope‐specific antibody to Val50 of AICD (IB 5 μg/ml) was a gift from Eli Lilly and Company and has been described before (Chavez‐Gutierrez et al, 2012). End‐specific antibodies to Aβ40, Aβ42, and Aβ43 characterized previously (Saito et al, 2011) were obtained from IBL (JP18580, JP18582, and JP 18583, immunohistochemistry (IHC) 2 μg/ml). Antibodies NT1 to the PS1 NTF (Covance SIG‐39194, IB 1:2,000), N1660 to NCT (Sigma N1660, IB 1:1,000), 22C11 to secreted soluble APP (Merck Millipore MAB348, IB 1:5,000), 4G8 to Aβ (Covance SIG‐39220, IP 1:500–1:2,500; and BioLegend 800701, IHC 1:500), Y188 to the APP C‐terminus (Abcam ab32136, IB 1:4,000), Penta‐His (Qiagen 34460, IB 1:2,000), and AT‐8 to hyperphosphorylated microtubule‐associated protein tau (Thermo Scientific MN1020, IHC 1:200) were obtained from the indicated companies. Rat monoclonal antibody 5E12 (IB 2.5 μg/ml) of IgG2a subclass was raised against residues 313‐333 (SKYNAESTERESQDTVAENDD) of human PS1.

Kretner B., Trambauer J., Fukumori A., Mielke J., Kuhn P., Kremmer E., Giese A., Lichtenthaler S.F., Haass C., Arzberger T, & Steiner H. (2016). Generation and deposition of Aβ43 by the virtually inactive presenilin‐1 L435F mutant contradicts the presenilin loss‐of‐function hypothesis of Alzheimer's disease. EMBO Molecular Medicine, 8(5), 458-465.

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Characterization of Alzheimer's Antibodies

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Antibodies to the PS1 CTF (3027 47, immunoblot (IB): 1:4,000), the PS2 CTF (BI.HF5c 23, IB: 1:2,000) as well as antibodies to total Aβ (3552 48, immunoprecipitation (IP) 1:500 and 2D8 49, IB 3 μg/ml) have been described previously. Monoclonal antibody 2G7 (IgG2b/k) (IB 3 μg/ml) to the PS1 NTF was raised in C57/BL6 mice against residues 39–52 (NDRRSLGHPEPLSN) of human PS1. Antibodies N1660 to NCT (Sigma N1660, IB 1:1,000), 22C11 to secreted soluble APP (Merck Millipore MAB348, IB 1:5,000), 4G8 to Aβ (Covance SIG‐39220, IP 1:500–1:2,500), Y188 to the APP C‐terminus (Abcam ab32136, IB 1:4,000), and Penta‐His (Qiagen 34460, IB 1:2,000) were obtained from the indicated companies. Species‐specific anti‐Aβ antibodies to Aβ40 (BAP24) and Aβ42 (BAP15) were kindly provided by Manfred Brockhaus (Roche Applied Science) and SULFO‐tagged according to the instructions of the supplier (Meso Scale Discovery (MSD)). The anti‐Aβ37 antibody (D2A6H) was obtained from Cell Signaling (#12467S) and SULFO‐tagged as above. The SULFO‐tagged antibody against Aβ38 was obtained from MSD.

Trambauer J., Rodríguez Sarmiento R.M., Fukumori A., Feederle R., Baumann K, & Steiner H. (2019). Aβ43‐producing PS1 FAD mutants cause altered substrate interactions and respond to γ‐secretase modulation. EMBO Reports, 21(1), e47996.

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