H3663

Manufactured by Merck Group

Sourced in United States, United Kingdom

H3663 is a laboratory equipment product. It is designed for specific laboratory functions. The core function of this product is to provide a controlled environment for various scientific experiments and analysis.

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Lab products found in correlation

F1804, by Merck Group (9 mentions) H6908, by Merck Group (5 mentions) Anti flag, by Merck Group (3 mentions) F3165, by Merck Group (3 mentions) M4439, by Merck Group (3 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions) Mitotracker red, by Thermo Fisher Scientific (3 mentions) Mg132, by Merck Group (2 mentions) A11122, by Thermo Fisher Scientific (2 mentions) Anti ha, by Merck Group (2 mentions) Dam1411288, by Merck Group (2 mentions) Anti mouse hrp igg peroxidase conjugate a9044, by Merck Group (2 mentions) Ab15246, by Cell Signaling Technology (2 mentions) P erk, by Cell Signaling Technology (2 mentions) Nitrocellulose membrane, by Cytiva (2 mentions) Bca assay, by Thermo Fisher Scientific (2 mentions) Bolt 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Hpa009975, by Merck Group (2 mentions) Ab9485, by Abcam (2 mentions)

64 protocols using h3663

Immunoprecipitation of Ku80, RNF113A, and Spindly

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For the Ku80 co-immunoprecipitation, the established stable cell lines were used to produce 1 mg WCE that was diluted to 0.25% NP-40 and pre-cleared for 30 min before incubation at 4 °C overnight with the primary anti-HA antibody (H3663, Sigma-Aldrich). Immunoprecipitated proteins were next isolated using Pierce^TM Protein G magnetic beads (ThermoFisher Scientific, Rockford, IL, USA) and washed thrice (Wash buffer composition: 60 mM KCl, 25 mM HEPES pH 7.9, 0.5 mM ETDA pH 8, 0.5% NP-40, 12% glycerol) before being analyzed by Western blotting.
For the RNF113A and Spindly co-immunoprecipitation, 500 μg of nuclear extracts were diluted to 0.1% NP-40 and 100 mM KCl, pre-cleared, then incubated with primary anti-HA antibody (H3663, Sigma-Aldrich) overnight. Immunoprecipitated proteins were isolated and washed the same way as for the Ku80 co-immunoprecipitation before analysis by Western blotting.

Abbasi S, & Schild-Poulter C. (2021). Identification of Ku70 Domain-Specific Interactors Using BioID2. Cells, 10(3), 646.

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Quantitative Western Blot Analysis Protocol

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Total proteins were extracted by RIPA buffer containing protease inhibitor as previously reported (Sun et al, 2016; Zhang et al, 2017). Samples were separated by SDS–PAGE and then transferred to PVDF membranes. After primary and secondary antibody incubation, protein expression was visualized using ECL Western Blotting Substrate (Thermo Fisher Scientific) and quantified using ImageJ software. The primary antibodies were anti‐Actin (1:2,000, A7811, Santa Cruz), anti‐α‐Actinin (1:1,000, A7811, Sigma‐Aldrich), anti‐Flag (1:1,000, F3165, Sigma‐Aldrich), anti‐GFP (1:1,000, A10262, Thermo Fisher Scientific), anti‐HA (1:1,000, H3663, Sigma‐Aldrich or 3724, Cell Signaling), anti‐HDAC9 (1:200, sc‐398003, Santa Cruz; Schroeder et al, 2018), anti‐MEF2a (1:200, sc‐17785, Santa Cruz; Reineke et al, 2014), anti‐NCoR1 (1:500, 5948, Cell Signaling; Simcox et al, 2015), and anti‐HDAC4 (1:500, ab12172, Abcam or 2072, Cell Signaling; Wein et al, 2016).

Li C., Sun X., Chen B., Zeng M., Du L., Liu T., Gu H., Liu Y., Li Y., Zhou L., Zheng X., Zhang Y., Zhang W., Liu Y., Shi C., Shao S., Shi X., Yi Y., Liu X., Wang J., Auwerx J., Wang Z.V., Jia F., Li R, & Duan S. (2019). Nuclear receptor corepressor 1 represses cardiac hypertrophy. EMBO Molecular Medicine, 11(11), e9127.

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Immunostaining of Nematode Muscle Proteins

Cited 1 time

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Adult and L1 larva nematodes were fixed and immunostained according to the method described by Nonet et al. (1993) (link) and described in further detail by Wilson et al. (2012a) (link). The following primary antibodies were used at 1:200 dilution: anti-UNC-89 (rabbit polyclonal EU30; Benian et al., 1996 (link)), anti-paramyosin (mouse monoclonal 5-23; Miller et al., 1983 (link)), anti–MHC A (mouse monoclonal 5-6; Miller et al., 1983 (link)), anti–MHC B (mouse monoclonal 5-8; Miller et al., 1983 (link)), anti–UNC-95 (rabbit polyclonal Benian-13; Qadota et al., 2007 (link)), anti-twitchin (rabbit polyclonal I I II; Benian et al., 1996 (link)), and anti-HA (mouse monoclonal; H3663; Sigma-Aldrich). The following antibodies were used at 1:100 dilution: anti-UNC-98 (rabbit polyclonal EU131; Mercer et al., 2003 (link)), and anti–CSN-5 (rabbit polyclonal; Miller et al., 2009 (link)). Secondary antibodies, also used at 1:200 dilution, included anti-rabbit Alexa 488 (Invitrogen, Thermo Fisher Scientific) and anti-mouse Alexa 594 (Invitrogen). Images were captured at room temperature with a Zeiss confocal system (LSM510) equipped with an Axiovert 100M microscope and an Apochromat ×63/1.4 numerical aperture oil immersion objective in ×2.5 zoom mode. The color balances of the images were adjusted by using Photoshop (Adobe, San Jose, CA).

Qadota H., Mayans O., Matsunaga Y., McMurry J.L., Wilson K.J., Kwon G.E., Stanford R., Deehan K., Tinley T.L., Ngwa V.M, & Benian G.M. (2016). The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle. Molecular Biology of the Cell, 27(10), 1606-1620.

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Ubiquitination Assay for Nrf2 Regulation

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Ubiquitination assay was performed as described elsewhere [20 (link)]. In brief, HEK293 cells (ATCC) were transfected with plasmids expressing HA-tagged Ub, V5-tagged Nrf2, and FLAG-tagged Keap1 [21 (link)] for 48 h and then treated with MGS for 16 h. Prior to lysis, cells were treated with 10 μM of MG132 (Sigma-Aldrich) for 3 h to block ubiquitin-dependent protein degradation. The total cell lysate was prepared by Pierce™ IP lysis buffer per the manufacturer’s protocols (Thermo Scientific). For precipitating V5-tagged Nrf2, 1 μg of anti-V5 antibody (R960–25, Thermo Scientific) was added to the cytosolic fraction. Immune complexes captured by protein A-sepharose (Thermo Scientific) were analyzed by immunoblotting for HA (H3663, Sigma) to reveal the ubiquitinated Nrf2.

Kim K.H., Lee J.Y., Ahn S., Won R., Kim S.J., Jeong S.I., Lee J.J., Kim J.I., Choi J.Y, & Joo M. (2020). The methanol extract of Guettarda speciosa Linn. Ameliorates acute lung injury in mice. BMC Complementary Medicine and Therapies, 20, 40.

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Western Blot Analysis of Drosophila Proteins

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Whole fly lysates were prepared by homogenizing the adult flies in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% [v/v] NP-40, 0.1% [w/v] SDS, 0.5% [w/v] sodium deoxycholate,1 mM EDTA, 5 mM DTT, and 0.5 mM PMSF). The homogenates were clarified by centrifugation at 21,000g at 4°C for 10 min, and the supernatant was used for Western blot. Ovary lysates and the immunoprecipitation samples were prepared as above. Mouse anti-HA (Sigma, H3663), rabbit anti-Tubulin (Sigma, T3526), rabbit anti-α-Tubulin (Abcam, ab52866), rat anti-Vasa (Developmental Studies Hybridoma Bank), mouse anti-Dicer-2 (Miyoshi et al. 2009 (link)), mouse anti-R2D2 (Nishida et al. 2013 (link)), mouse anti-Ago2 (Miyoshi et al. 2005 (link)) (kind gifts from Dr. Mikiko Siomi and Dr. Haruhiko Siomi), and rabbit anti-R2D2 (Liu et al. 2003 (link)) (kind gift from Dr. Qinghua Liu), were used as primary antibodies. IRDye 800CW goat anti-mouse IgG, IRDye 800CW goat anti-rat IgG, IRDye 800CW goat anti-rabbit IgG, and IRDye 680RD goat anti-rabbit IgG (Licor) were used as secondary antibodies. The membrane was scanned on an Odyssey imaging system (Licor).

Kandasamy S.K., Zhu L, & Fukunaga R. (2017). The C-terminal dsRNA-binding domain of Drosophila Dicer-2 is crucial for efficient and high-fidelity production of siRNA and loading of siRNA to Argonaute2. RNA, 23(7), 1139-1153.

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Ultrastructural and Protein Localization

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The experiments are performed as our previous described procedures. Thirty-nine EM images were obtained from thin sections using a JEM1400 electron microscope (JEOL, Akishima, Tokyo, Japan). The relative cytosolic areas in cross-sections of 10 cells were analyzed by Image-Pro Plus software. And fluorescence signals obtained using anti-FLAG (mouse) (A8592, Sigma), anti-FLAG (rabbit) (20543-1-AP, Proteintech), anti-HA (mouse) (H3663, Sigma), anti-HA (rabbit) (C29F4, Cellular signaling), anti-SC35 antibody (Ab11826, Abcam), and anti-SF2 antibody (sc-33652, Santa Cruz) were acquired and analyzed by Carl Zeiss LSM 880 microscope (Carl Zeiss, Germany). At least 10 cells from each group were analyzed.

Chen Z.H., Chen T.Q., Zeng Z.C., Wang D., Han C., Sun Y.M., Huang W., Sun L.Y., Fang K., Chen Y.Q., Luo X.Q, & Wang W.T. (2020). Nuclear export of chimeric mRNAs depends on an lncRNA-triggered autoregulatory loop in blood malignancies. Cell Death & Disease, 11(7), 566.

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Antibody Selection for HIV-1 Vif Detection

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The antibodies used were as follows: anti-HIV1 Vif antibody [319] (ab66643, Abcam); mouse anti-HA antibody [HA-7] (H3663, Sigma-Aldrich); rabbit anti-HA antibody (H6908, Sigma-Aldrich); anti-beta-actin antibody (cw0096a, CWBIO); MYC-tag antibody [2D11A8] (66004-1-Ig, Proteintech Group, Manchester, UK); anti-FLAG M2 antibody (F1804, Sigma-Aldrich); anti-CBFb antibody [EPR6322] (ab133600, Abcam); mouse anti-p24 antibody (produced in-house); anti-mouse IgG (H + L) antibody (4741806, KPL); anti-rabbit IgG (H + L) antibody (0741506, KPL).

Chen H., Zhang R., Luo R.H., Yang L.M., Wang R.R., Hao X.J, & Zheng Y.T. (2017). Anti-HIV Activities and Mechanism of 12-O-Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(9), 1498.

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Detailed Western Blot and Immunohistochemistry Protocols for Drosophila and Cell Lines

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For doing western blots in Drosophila, the following primary antibodies were used: anti-FUS (Bethyl Laboratories; A300-302A, 1:2000), anti-MBNL1 (Millipore; MabE70, 1:2000), and anti-mbnl1 (Invitrogen; MA1-16967, 1:1000). DyLight fluorescent secondary antibodies (Thermo Fisher Scientific) were used at a concentration of 1:10,000
For the Drosophila NMJ analysis, the following primary antibodies were used: Alexa Fluor 488-conjugated goat anti-HRP (Jackson Immuno Research; 123-545-021, 1:200) and mouse anti-DLG 4F3 (DSHB, 1:100). The following secondary antibodies were used: Alexa Fluor 647-conjugated anti-Phalloidin (Invitrogen; A22287, 1:250) and goat antimouse Alexa Fluor 546 (Invitrogen, A11030, 1:500).
The following primary antibodies were used: anti-G3BP1 (Protein Tech; 13057-2-AP, 1:2000) and anti-HA7 (Sigma, H3663, 1:500). Alexa-Fluor fluorescent secondary antibodies (Invitrogen) were used at a concentration of 1:500 for doing Western blots in HEK293 and N2a cell lines.
The following primary antibodies were used for doing immunoblotting in primary neuronal cells: anti-MAP2 (EMD Millipore; AB5622, 1:500), anti-FUS (Proteintech; 11570-1-AP, 1:200), anti-TIA1 (Santa Cruz; SC-1751, 1:250), anti-G3BP1 (Proteintech; D5444, 1:100), anti-MBNL1 (Millipore; MABE70, 1:100), and anti-NEFL (Novus Biologicals; NB300-222, 1:300).

Casci I., Krishnamurthy K., Kour S., Tripathy V., Ramesh N., Anderson E.N., Marrone L., Grant R.A., Oliver S., Gochenaur L., Patel K., Sterneckert J., Gleixner A.M., Donnelly C.J., Ruepp M.D., Sini A.M., Zuccaro E., Pennuto M., Pasinelli P, & Pandey U.B. (2019). Muscleblind acts as a modifier of FUS toxicity by modulating stress granule dynamics and SMN localization. Nature Communications, 10, 5583.

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Ebf1 Binding Site Identification

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ChIP was performed using the EZ-ChIP kit (Millipore; 17-371) according to the manufacturer's instruction. In brief, chromatin was isolated from mouse neuroblastoma N2a cells stably overexpressing Ebf1-tagged with HA or empty vector either in basal condition or challenged with LPS (1 µg/ml, 2 h). Monoclonal anti-HA antibody produced in mouse (Sigma-Aldrich, H3663) and anti-RNA Pol II antibody were used for IP. The promoter sequences corresponding to the upstream of the start site of Rgs5 were used for analysis. Primer sets corresponding to the regions are as follows: − 2128 to − 2057 bp from TSS, forward 5′-TCACATGAACTCCTTTGGGACA-3′, reverse 5′-ATGGAGGGTGATGTCTTGGC-3′; − 1678 to − 1580 bp from TSS, forward 5′-TAGCTAGGCATGGTAGCAAG-3′, reverse 5′-ATAGCTCACTGCATCATCAA-3′; − 1973 to − 1828 bp from TSS, forward 5′-TGTCCCAAGGAGTCTCTTCT-3′, reverse 5′-ACAAACTGTCCAAGTTCCGC-3′.

Yin S., Ma X.Y., Sun Y.F., Yin Y.Q., Long Y., Zhao C.L., Ma J.W., Li S., Hu Y., Li M.T., Hu G, & Zhou J.W. (2023). RGS5 augments astrocyte activation and facilitates neuroinflammation via TNF signaling. Journal of Neuroinflammation, 20, 203.

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Circadian Neuronal Protein Quantification

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Whole-mount immunohistochemistry of fly brains was done as previously described [16 (link)]. For PDF staining, adult flies were entrained to LD for four days and dissected at ZT2 or 14. For PER staining, flies were entrained to LD for four days and then released into DD. Brains were dissected on the second day of DD at six time points. Mouse anti-PDF (1:400, DSHB), rabbit anti-GFP (1:600, ThermoFisher A-6455), rabbit anti-PER (1:1500, gift from Dr. R. Stanewsky), mouse anti-HA (1:400, Sigma H3663), and rabbit anti-HA (1:100, Sigma H6908) antibodies were used. All samples were imaged using a Leica TCS SP8 confocal microscope with a constant laser setting for each time point. In total, 8–10 brains for each genotype were dissected for imaging. ImageJ software [62 (link)] was used for GFP, PER, and PDF quantification from at least five brains. For quantification, the average signals of three neighboring background areas were subtracted from the signal intensity in each circadian neuron. Each experiment was conducted three times.

Nian X., Chen W., Bai W., Zhao Z, & Zhang Y. (2019). miR-263b Controls Circadian Behavior and the Structural Plasticity of Pacemaker Neurons by Regulating the LIM-Only Protein Beadex. Cells, 8(8), 923.

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