Nupage mops running buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States

NuPAGE MOPS running buffer is a ready-to-use buffer solution designed for use with the NuPAGE electrophoresis system. It is used to maintain the pH and ionic conditions during the separation of proteins by SDS-PAGE.

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30 protocols using nupage mops running buffer

Quantifying Apoptosis-Inducing Factor Levels

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Whole cell lysates of fibroblasts and transdifferentiated osteoblasts were subjected to electrophoresis for 50 min at 200 V on NuPage 4–12% BIS-TRIS gel with NuPAGE MOPS Running Buffer (Invitrogen). After protein transfer and blocking, the nitrocellulose membrane was incubated with primary antibodies for AIFM1 (Abcam; cat no. AB1998) and actin (Abcam; cat no. AB 14128) overnight at 4 °C. After 1 h incubation with secondary antibody IRDye 800 CW goat anti-rabbit IgG and the IRDye 680 CW goat anti-mouse IgG antibodies (LICOR Bioscience), the NC membrane was scanned, analyzed, and quantified with Odyssey Infrared Imaging system equipped with the Odyssey v3.0 software (LICOR Bioscience). Additional details are available in supplemental methods.

Miyake N., Wolf N.I., Cayami F.K., Crawford J., Bley A., Bulas D., Conant A., Bent S.J., Gripp K.W., Hahn A., Humphray S., Kimura-Ohba S., Kingsbury Z., Lajoie B.R., Lal D., Micha D., Pizzino A., Sinke R.J., Sival D., Stolte-Dijkstra I., Superti-Furga A., Ulrick N., Taft R.J., Ogata T., Ozono K., Matsumoto N., Neubauer B.A., Simons C, & Vanderver A. (2017). X-linked hypomyelination with spondylometaphyseal dysplasia (H-SMD) associated with mutations in AIFM1. Neurogenetics, 18(4), 185-194.

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Extracting Parasite Proteins for SDS-PAGE

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Cultures of late-stage parasites (35–45 hpi) were first treated with 0.05% saponin and washed twice with PBS to enrich for parasites. The pellet was then resuspended in 1 ml of 0.5% Triton X and spun down hard. The supernatant was discarded and the pellet was resuspended in 2% SDS before being spun down hard again. The SDS extract was then used for SDS-PAGE while the pellet was discarded. The SDS extracts were mixed with 4× NuPAGE LDS loading buffer (Invitrogen) and NuPAGE sample reducing agent (Invitrogen) in 1:15 v/v ratio, heated at 100 °C for 10 min, cooled and loaded into the wells of the pre-cast NuPAGE Novex 4–12% Bid-Tris gels (Invitrogen). NuPAGE MOPS running buffer (Invitrogen) was used, as well as pre-stain plus (Fermentas) protein ladder.

Ch’ng J.H., Sirel M., Zandian A., del Pilar Quintana M., Chun Leung Chan S., Moll K., Tellgren-Roth A., Nilsson I., Nilsson P., Qundos U, & Wahlgren M. (2017). Epitopes of anti-RIFIN antibodies and characterization of rif-expressing Plasmodium falciparum parasites by RNA sequencing. Scientific Reports, 7, 43190.

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SDS-PAGE and Western Blotting Assay

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Fifteen micrograms of nuclear extract was diluted to 30 μl with H₂O, 10 μl of 4 × loading buffer (50% (v/v) glycerol, 0.16 M Tris-HCl (pH 6.8), 2% (w/v) SDS, 0.01% (w/v) bromophenol blue, and 350 mM DTT) was added, and the samples were denatured at 95 °C for 15 min. Total protein of 7.5 μg and 5 μl of Precision Plus Protein Dual Color Standard (Bio-Rad Laboratories, Hercules, CA, USA) were loaded onto a NuPAGE 10% Bis-Tris gel. SDS-PAGE was performed in 1 × NuPAGE MOPS running buffer (Invitrogen) at 100 V for 2 h. After electrophoresis, proteins were blotted onto a PVDF membrane (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) using 1 × NuPAGE Transfer buffer (Invitrogen) with 20% methanol overnight at 4 °C at 100 mA. Blocking and incubation with primary (1:1000 dilutions) and secondary antibodies (donkey anti-rabbit AlexaFlour 488 and donkey anti-goat AlexaFluor 488; Invitrogen) was performed as described previously. Fluorescence signals were visualized using a Typhoon Trio Variable Mode Imager (GE, Fairfield, CT, USA).

Eggers S., Smith K.R., Bahlo M., Looijenga L.H., Drop S.L., Juniarto Z.A., Harley V.R., Koopman P., Faradz S.M, & Sinclair A.H. (2014). Whole exome sequencing combined with linkage analysis identifies a novel 3 bp deletion in NR5A1. European Journal of Human Genetics, 23(4), 486-493.

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Purification of Recombinant Proteins from E. coli

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Overnight E. coli cultures grown at 37 °C were diluted 1:50 with LB medium, grown to OD₆₀₀ of 0.5–0.8, induced by adding isopropyl-β-D-thiogalactoside (IPTG) to 1 mM and incubated for further 2 h. Large scale preparation was carried out using 100 ml of induced cultures. Bacteria were pelleted by centrifugation and resuspended in CellLytic B reagent (Sigma). After centrifugation the soluble fraction of the lysate was used for protein purification using HisSelect™ Spin Columns (Sigma) according to manufacturer’s protocol. The fractions were analyzed on 12 % NuPAGE™ Novex Bis-Tris SDS–PAGE gels.
The gels were run using NuPAGE™ MOPS running buffer (Invitrogen) at 200 V for 1 h and stained with Coomassie Blue.

Karlyshev A.V., Thacker G., Jones M.A., Clements M.O, & Wren B.W. (2014). Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation. FEBS Open Bio, 4, 468-472.

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SDS-PAGE and Western Blot Analysis of Recombinant Thioredoxin

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rhTrx1 (DTT-treated or non-treated) was mixed with NuPage LDS sample buffer (Invitrogen, Carlsbad, CA, USA), kept at 70°C for 10 min and resolved under non-reducing conditions on NuPAGE 4–12% gradient Bis-Tris gels (Invitrogen) in a XCELL II Electrophoresis cell (Novex, San Diego, CA, USA) using NuPage MOPS running buffer (Invitrogen). A pre-stained standard (SeeBlue Plus 2; Invitrogen) was included as reference. For SDS-PAGE analysis, 5 µg Trx1 was loaded per lane and the gel was subsequently stained with Simply Blue Safestain (Invitrogen). For Western blot analysis, 1 µg Trx1 was loaded per lane and the proteins were transferred to 0.2 µm pore size nitrocellulose membrane (Invitrogen) after separation using a MiniTrans-Blot apparatus (Bio-Rad, Hercules, CA, USA) with 20 mM Tris pH 8.6. Membranes were blocked for 1 h at room temperature with 4% fetal calf serum (FCS) in PBS. After washing with PBS, the membranes were incubated with 1 µg/ml of biotinylated mAb 2G11 or pAb in PBS with 0.5% FCS. The membranes were washed and incubated for 1 h at room temperature with streptavidin-alkaline phosphatase (Mabtech) diluted 1∶1000 in PBS, washed again and developed with BCIP/NBT Plus (Mabtech) for 5 min before being rinsed in tap water.

Lundberg M., Curbo S., Reiser K., Masterman T., Braesch-Andersen S., Areström I, & Ahlborg N. (2014). Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid. PLoS ONE, 9(7), e103554.

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Dephosphorylation of Csm3/Tof1 by λ-PP

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Prior to electrophoresis Csm3/Tof1 was treated with Lambda protein phosphatase (λ-PP) in a reaction (100 μl) containing 50 mM HEPES-NaOH pH 7.5, 100 mM NaCl, 2 mM DTT, 1 mM MnCl₂, 300 nM Csm3/Tof1 and 0.1 mg/ml λ-PP (Zhuo et al., 1993 (link)) for 40 min at 37°C. Samples (10 μl) were separated through 5% polyacrylamide gels containing 353 mM Bis-Tris-HCL pH 6.8, 100 μM ZnCl₂ and 50 μM Phosbind (APExBio). Control gels were also run in the absence of Phosbind. Electrophoresis was performed in 1x NuPAGE MOPS running buffer (Invitrogen) at 30 mA for ∼90 min and gels were stained with Coomassie InstantBlue (Expedeon).

Baretić D., Jenkyn-Bedford M., Aria V., Cannone G., Skehel M, & Yeeles J.T. (2020). Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork. Molecular Cell, 78(5), 926-940.e13.

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Western Blot Protein Analysis Protocol

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Forelimb and hindlimb tendons were dissected, taking care to avoid enthesis and muscle insertions. Tissues were lysed in RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.1% SDS, 1.0% NP-40, 0.5% sodium deoxycholate) containing COMPLETE Protease Inhibitor cocktail (Roche). Protein quantification of cell and tissue lysates was performed using the BioRad DC Protein assay. Equal amounts of total protein were prepared in LDS sample buffer (Invitrogen) and 100mM DTT and run on 4–12% NuPAGE Bis-Tris gels (Invitrogen) in NuPAGE MOPS running buffer (Invitrogen). Wet transfer with NuPAGE transfer buffer (Invitrogen)/20% methanol was used to transfer protein onto Millipore Immobilon P (PVDF) membranes. Blots were washed thrice with TBS-0.1%Tween-20 and blocked in 5% serum for 1 hour. Blots were incubated in primary antibody overnight before being incubated in HRP-conjugated secondary antibody for one hour at room temperature. Blots were developed with ECL Substrate (BioRad) and imaged using the ChemiDoc XRS+ System (BioRad) for detection.

Schlesinger S., Seo S., Pryce B., Tufa S.F., Keene D.R., Huang A.H, & Schweitzer R. (2020). Loss of Smad4 in the Scleraxis Cell Lineage Results in Postnatal Joint Contracture. Developmental biology, 470, 108-120.

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SDS-PAGE Protein Analysis Protocol

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After boiled for 5 min, 5 µg of total protein mixed with 4X sample buffer (Invitrogen, cat. # NP0007) was loaded on 4–12% NuPAGE Bis-Tris gel (Invitrogen, Cat. # NP0321BOX) with NuPAGE MOPS running buffer (Invitrogen, Cat. # NP0001). SeeBlue Plus2 Protein Ladder (Invitrogen, cat. # LC5925) or BenchMark Protein Ladder (Invitrogen, Cat. # 10747-012) was used to estimate the protein size. Gel was stained with SimpleBlue SafeStain (Invitrogen, Cat. # LC6060) for overnight at 4°C and destained with HPLC water. The gel image was taken by Bio-Rad ChemiDoc XRS+ Imaging System.

Gounarides J., Cobb J.S., Zhou J., Cook F., Yang X., Yin H., Meredith E., Rao C., Huang Q., Xu Y., Anderson K., De Erkenez A., Liao S.M., Crowley M., Buchanan N., Poor S., Qiu Y., Fassbender E., Shen S., Woolfenden A., Jensen A., Cepeda R., Etemad-Gilbertson B., Giza S., Mogi M., Jaffee B, & Azarian S. (2014). Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis. PLoS ONE, 9(10), e111472.

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Quantification of GRP78 Protein Levels

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HaCat cells were plated at a concentration of 3.5 x10 5 cells per well in 6-well plates and left for 24 h. Media was removed, cells were washed in PBS and exposed to media containing different concentrations of biocides or ILs for 24 h. Cells were washed with PBS and lysed on ice in buffer containing 50 mM Tris-HCl, 20 mM EDTA and 1% nonidet P-40 for 15 minutes. Samples were centrifuged for 15 min at 14,500 rpm and the supernatant retained. Equal loading of protein samples on SDS-PAGE was determined by bicinchoninic acid (BCA) assay (Thermo Scientific Pierce, Leicestershire, UK). Whole cell lysates were separated on reduced 4-12% Bis-Tris NuPAGE gels using NuPAGE MOPS running buffer (Invitrogen). After blotting to PVDF membrane (GE Healthcare, Buckinghamshire, UK), blotted membranes were blocked with 5% non-fat dry milk in PBS for 1 h and probed for GRP78 with mouse α-KDEL (Stressgen, British Columbia, Canada), mouse α-γ-tubulin (Sigma) and HRP-conjugated goat α-mouse secondary (Jackson Immunoresearch, PA, USA).
Antibody binding was detected using immobilon western substrate (Millipore, UK) according to the manufacturer's instructions.

McLaughlin M., Gilea M.A., Earle M.J., Seddon K.R., Gilmore B.F, & Kelly S.A. (2021). Characterization of ionic liquid cytotoxicity mechanisms in human keratinocytes compared with conventional biocides. Chemosphere, 270.

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Western Blot Protein Analysis

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Cells were washed with PBS and lysed with lysis buffer (10 mM Tris-HCl (pH 8), 150 mM NaCl, 1% Triton X100 and complete protease inhibitor mixture (Roche)). Total protein extracts were run on 4–12% NuPAGE Bis-Tris gels in NuPAGE MOPS running buffer (Thermo Fisher), transferred to nitrocellulose membranes (Amersham Biosciences) and analyzed using primary antibodies. Primary antibodies were incubated overnight at 4°C and secondary HRP conjugated antibodies (Jackson ImmunoResearch, 1:10,000) were incubated for 1 hour at room temperature. Thermo Pierce ECL detection reagents were used. See Supplemental Experimental Procedures for full list of antibodies used.

Conway A.E., Van Nostrand E.L., Pratt G.A., Aigner S., Wilbert M.L., Sundararaman B., Freese P., Lambert N.J., Sathe S., Liang T.Y., Essex A., Landais S., Burge C.B., Jones D.L, & Yeo G.W. (2016). Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival. Cell reports, 15(3), 666-679.

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