Mw25000

Manufactured by Polysciences

Sourced in Germany, United States, Netherlands

The MW25000 is a laboratory equipment product designed for molecular weight determination. It is a versatile instrument that can be used to measure the molecular weight of various substances, including polymers, proteins, and other macromolecules. The core function of the MW25000 is to provide accurate and reliable measurements of molecular weight using advanced techniques such as light scattering or size exclusion chromatography.

Automatically generated - may contain errors

Lab products found in correlation

Hek293t cell, by American Type Culture Collection (2 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Anti rabbit, by Merck Group (1 mentions) Hek293ft cells, by American Type Culture Collection (1 mentions) Anti mouse, by Merck Group (1 mentions) Anti ha, by Cell Signaling Technology (1 mentions) Anti β actin, by Cell Signaling Technology (1 mentions) Anti flag agarose, by Merck Group (1 mentions) Anti ha c29f4, by Cell Signaling Technology (1 mentions) Anti flag m2, by Merck Group (1 mentions) F3917, by Merck Group (1 mentions) Ha antibody, by Merck Group (1 mentions) Flag antibody, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Dual luciferase assay system, by Promega (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Protein a agarose beads, by GE Healthcare (1 mentions) Freestyle 293 f, by Thermo Fisher Scientific (1 mentions)

11 protocols using mw25000

Investigating p62 and ATF2 Interaction

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HEK293FT cells (ATCC, Manassas, VA, USA; #CRL-3216) or HEK293T cells (ATCC, Manassas, VA, USA; #CRL-1573) were cultured in 6-well plate and transfected with 1.5 μg Flag-p62 and 1.5 μg HA-ATF2 plasmid per well by polyethylenimine (linear, MW-25000, Polysciences, Hirschberg, Germany) for 24 h. Isoproterenol (10 μM) was added half an hour before the cells were collected. The protein lysate of 1500 μg was incubated with anti-Flag-agarose (Sigma-Aldrich, Munich, Germany) overnight for immunoprecipitation, together with 50 μg lysate as input to be loaded for western blotting detection of HA (anti-HA, Cell Signaling Technology) or Flag tag (anti-Flag). Antibodies were obtained from the following sources: anti-HA (C29F4) (Cell Signaling, Danvers, MA, USA; #3724S; dilution: 1:1000), anti-FLAG-M2 (Sigma-Aldrich, St. Louis, MO, USA; #F1804; dilution: 1:2000), anti-beta-actin (Cell Signaling, Danvers, MA, USA #4967S; dilution: 1:2000), anti-rabbit (Sigma-Aldrich, St. Louis, MO, USA; #A3687; dilution: 1:20,000), anti-mouse (Sigma-Aldrich, St. Louis, MO, USA; #A3562; dilution: 1:20,000).

Fischer K., Fenzl A., Liu D., Dyar K.A., Kleinert M., Brielmeier M., Clemmensen C., Fedl A., Finan B., Gessner A., Jastroch M., Huang J., Keipert S., Klingenspor M., Brüning J.C., Kneilling M., Maier F.C., Othman A.E., Pichler B.J., Pramme-Steinwachs I., Sachs S., Scheideler A., Thaiss W.M., Uhlenhaut H., Ussar S., Woods S.C., Zorn J., Stemmer K., Collins S., Diaz-Meco M., Moscat J., Tschöp M.H, & Müller T.D. (2020). The scaffold protein p62 regulates adaptive thermogenesis through ATF2 nuclear target activation. Nature Communications, 11, 2306.

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Lentiviral Vector Production Protocol

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6 μg amounts of lentiviral plasmids (pUltra-eGFP, pUltra-eGFP-P2A-Cre, pUltra-eGFP-P2A-Cre-T2A-Impact) were transfected together with 3 μg of GAG-Pol and VSVG-Rev plasmids into 293T cells in 10 cm plates using polyethyleneimine (MW25000, Polyscience Inc. #23966). 10 μM forskolin (SIGMA #F3917) was added to the culture 16 h after transfection, and supernatant was collected at 48 h after transfection. Pooled viral supernatant was centrifuged and concentrated.

Tajima Y., Ito K., Yuan Y., Frank M.O., Saito Y, & Darnell R.B. (2023). NOVA1 acts on Impact to regulate hypothalamic function and translation in inhibitory neurons. Cell reports, 42(2), 112050.

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Ahr-ARNT Dimerization Detection

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Plasmids were generated as described (Supplemental Information) and polyethylenimine (MW25000; Polysciences) were mixed at the ratio of 1:4 for 20 minutes at room temperature before adding into HEK293T cell culture medium. 24 hours later, transfected HEK293T cells were lysed in lysis buffer (1% TritonX-100, 150 mM NaCl, 20 mM HEPES [pH7.5], 1 mM EDTA) containing protease inhibitor cocktails (Roche). The lysates were immunoprecipitated with corresponding antibodies at 4°C for 3 hours and subjected to SDS-PAGE and immunobloting with Flag antibody (Sigma), HA antibody (Sigma), Ahr antibody (Novus), or ARNT antibody (Novus). To detect the dimerization between Ahr and ARNT, transfected cells were treated with or without 200 nM FICZ (Enzo) for 3 hours prior to the immunoprecipitation.

Li S., Heller J.J., Bostick J.W., Lee A., Schjerven H., Kastner P., Chan S., Chen Z.E, & Zhou L. (2016). Ikaros Inhibits Group 3 Innate Lymphoid Cell Development and Function by Suppressing the Aryl Hydrocarbon Receptor Pathway. Immunity, 45(1), 185-197.

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TP53 Reporter Assay in HEK293 Cells

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HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and a 100 U/ml penicillin-streptomycin mixed solution. Cells were seeded into 35 mm-diameter plates and transfected with the expression plasmids and TP53 reporter plasmids using polyethylenimine MW 25000 (Polysciences, Warrington, PA, USA). After 24 h, firefly and Renilla luciferase activities were determined with the Promega Dual Luciferase Assay System (Promega, Madison, WI, USA). The pRL-EF vector, which expresses Renilla luciferase under the control of the EF-1α promoter, was used to normalize the transfection efficiency of the luciferase reporters. The values shown are the averages of experiments repeated three times, with each transfection performed in duplicate for each experiment.

Haraoka Y., Akieda Y., Nagai Y., Mogi C, & Ishitani T. (2022). Zebrafish imaging reveals TP53 mutation switching oncogene-induced senescence from suppressor to driver in primary tumorigenesis. Nature Communications, 13, 1417.

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Cell Culture and Transfection Protocols

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HEK293T and HeLa cells (from ATCC) were cultured in DMEM (Gibco) supplemented with 10% FBS (Hyclone), non-essential amino acids (Gibco) and 2-mercaptoethanol (Pierce). HeLa cells were transfected with plasmids using Lipofectamine 2000 (Invitrogen) under the instruction of manufacturers. HEK293T cells were transfected using PEI (MW-25000, Polysciences).

Ding X., Jiang W., Zhou P., Liu L., Wan X., Yuan X., Wang X., Chen M., Chen J., Yang J., Kong C., Li B., Peng C., Wong C.C., Hou F, & Zhang Y. (2015). Mixed Lineage Leukemia 5 (MLL5) Protein Stability Is Cooperatively Regulated by O-GlcNac Transferase (OGT) and Ubiquitin Specific Protease 7 (USP7). PLoS ONE, 10(12), e0145023.

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Expression and Purification of IgG 8H9

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The anti-B7-H3 IgG 8H9 was expressed and purified according to the instruction as previously described (Li D. Z. et al., 2018 (link)). Briefly, the IgG 8H9 was expressed by transient transfection of the plasmids with polythyleneimine (MW25000, Polyscience) in Free-style 293-F (Invitrogen) cells. The expressed IgG proteins were purified by protein A agarose beads (GE Healthcare).

Yang S., Cao B., Zhou G., Zhu L., Wang L., Zhang L., Kwok H.F., Zhang Z, & Zhao Q. (2020). Targeting B7-H3 Immune Checkpoint With Chimeric Antigen Receptor-Engineered Natural Killer Cells Exhibits Potent Cytotoxicity Against Non-Small Cell Lung Cancer. Frontiers in Pharmacology, 11, 1089.

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Luciferase Reporter Assay for Nose Keratinocytes

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Reporter gene assays were performed with some adaptations (Williamson et al., 2006 (link)) using the Dual Luciferase Reporter Assay System (Promega; E1919) as indicated by the manufacturer. Nose keratinocytes at passage 6 (Control 2; HNPK 1) or 15 (Controls 1–3; HNPK 1 or 2) were seeded in two independent experiments in triplicates at 30,000 cells/cm² in 96-well plates and cultured in 1/3 CnT-9 and 2/3 CnT-07 with 1X antibiotics/antimycotics (CELLnTEC). 24 h after seeding, cells were transiently transfected (Williamson et al., 2006 (link)). Briefly, keratinocytes were transfected with 1.5 µl polyethylenimine (1 mg/ml; linear; Polyscience Inc.; MW-25000), 0.2 µg TOP or FOP reporter plasmid (a kind gift of Prof. Hans Clevers, Hubrecht Institute, KNAW, Utrecht, Netherlands), and 7 ng plasmid encoding the Renilla Luciferase for normalization. Cells were incubated 2 h at 35°C with shaking every 15 min. Transfection medium was replaced by 1/3 CnT-09 and 2/3 CnT-07 with 1X antibiotics/antimycotics (CELLnTEC) and 10% FCS and cultured for 24 h. Measurements were performed with a TEKAN infinite 200, with default parameters for luminescence measurement.

Balmer P., Hariton W.V., Sayar B.S., Jagannathan V., Galichet A., Leeb T., Roosje P, & Müller E.J. (2021). SUV39H2 epigenetic silencing controls fate conversion of epidermal stem and progenitor cells. The Journal of Cell Biology, 220(4), e201908178.

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Transfection of HEK293T cells with Plasmids

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HEK293T cells (ATCC) were maintained in Dulbecco’s modified Eagles medium (Corning) with 10% fetal bovine serum (Sigma) and penicillin/streptomycin (Sigma). This cell line was tested and found negative for mycoplasma contamination. Cells growing at 50–70% confluence were transiently transfected using linear polyethylenimine (M.W. 25000, Polysciences) as described (Ossipova et al., 2009 (link)). Each 60 mm dish of cells received 1 µg of pCS2 plasmids encoding FLAG-Pk3, FLAG-GFP-Pk3∆PET and Myc-Par3 constructs. For transfections, pCS2 was added to plasmid DNA mixture to reach the total DNA amount of 3 µg.

Chuykin I., Ossipova O, & Sokol S.Y. (2018). Par3 interacts with Prickle3 to generate apical PCP complexes in the vertebrate neural plate. eLife, 7, e37881.

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Recombinant RSV N Protein Production

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The RSV N protein (GenBank accession AY911262.1) was cloned into the pCAG-OSF vector with two N-terminal strep-tag II between xho I and kpn I by ClonExpress^® II one step cloning kit (Vazyme). This plasmid was transfected into HEK293F cells (2 × 10⁶) with polyethylenimine linear (PEI, MW25000, Polysciences, cat: 23966-1). Protein expression was performed in 1 L erlenmeyer flasks with 300 mL of expression medium (Gibco, FreeStyle^TM 293 expression medium, cat: 2446428) at 37 °C, 5% CO₂ and 130 rpm for 3 days. The cells were harvested by centrifugation at 3000 × g for 10 min at 4 °C and subsequently washed with phosphate buffered saline (PBS). Following a second centrifugation step, the cells were frozen and stored at −80 °C until purification.

Wang Y., Zhang C., Luo Y., Ling X., Luo B., Jia G., Su D., Dong H, & Su Z. (2023). Cryo-EM structure of the nucleocapsid-like assembly of respiratory syncytial virus. Signal Transduction and Targeted Therapy, 8, 323.

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Lentiviral expression of mutant Med1

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Lentiviral expression constructs were generated using pUltra plasmids (Addgene #24129). For lentivirus packaging, psPAX2 (Addgene #12260) and pMD2.G (Addgene #12259) plasmids were used. 6 μg amounts of lentiviral plasmids carrying mouse Med1 cDNA with mutation in the IDR (pUltra-MED1, pUltra-MED1(EDtoA) pUltra-MED1(RHKtoA)) were transfected together with 3 μg of GAG-Pol and VSVG-Rev plasmids into 293T cells in 10 cm plates using polyethyleneimine (MW25000, Polyscience Inc. #23966). 10μM forskolin (SIGMA #F3917) was added to the culture 16 hours after transfection, and supernatant was collected at 48 and 72 hours after transfection. Pooled viral supernatant was centrifuged and concentrated.

Lyons H., Veettil R.T., Pradhan P., Fornero C., De La Cruz N., Ito K., Eppert M., Roeder R.G, & Sabari B.R. (2023). Functional partitioning of transcriptional regulators by patterned charge blocks. Cell, 186(2), 327-345.e28.

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