Nupage sample buffer

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

NuPAGE sample buffer is a concentrated solution designed for sample preparation in protein electrophoresis. It is used to denature and prepare protein samples for analysis on NuPAGE electrophoresis gels.

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NuPAGE (743 citations) Sample buffer (138 citations) NuPAGE SDS sample buffer (13 citations) NuPAGE LDS sample loading buffer (11 citations) Novex NuPage LDS sample buffer (7 citations) NuPAGE sample loading buffer (7 citations) NuPAGE LDS Sample Buffer and Reducing Agent (6 citations) NuPAGE LDS electrophoresis sample buffer (5 citations) NuPAGE lithium dodecyl sulphate (LDS) sample buffer (3 citations) NuPAGE LDS Sample Buffer (4×) buffer (3 citations)

109 protocols using nupage sample buffer

Protein Extraction and Immunoblotting

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The samples were lysed in NuPAGE Sample Buffer (Invitrogen),
sonicated and boiled. Samples were resolved by 4%–12% Bis-Tris
Protein Gels and blotted onto a PVDF membrane. Western blotting was
performed according to standard procedures.

Huang J., Zhang J., Bellani M.A., Pokharel D., Gichimu J., James R.C., Gali H., Ling C., Yan Z., Xu D., Chen J., Meetei A.R., Li L., Wang W, & Seidman M.M. (2019). Remodeling of Interstrand Crosslink Proximal Replisomes Is Dependent on ATR, FANCM, and FANCD2. Cell reports, 27(6), 1794-1808.e5.

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Optimized Apoptosis Assay Protocols

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Auranofin, A23187 and BAPTA-AM were from Tocris (R & D Systems, Abingdon, UK), Q-VD-OPh (non-O-methylated) was from Calbiochem (Merck Millipore, Nottingham, UK). CM-H₂DCFCA and Nu-PAGE sample buffer were from Invitrogen (Life Technologies, Paisley, UK). Anti-caspase-3 and anti-gelsolin antibodies were from Cell Signaling Technology (Beverley, MA, USA). Annexin V-FITC was from Abcam (Cambridge, UK). Fluo-2 LR-AM (acetoxymethylester) was from Teflabs (Cambridge Bioscience, Cambridge, UK). The lactate dehydrogenase (LDH) assay kit was from BioVision inc. (Milpitas, CA, USA). All other reagents, including the TRXR assay kit (CS0170), were from Sigma (Poole, UK), and were of analytical grade.

, & Harper M.T. (2017). Auranofin, a thioredoxin reductase inhibitor, causes platelet death through calcium overload. Platelets, 30(1), 98-104.

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Western Blot Analysis of RNA-Binding Proteins

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Western blot analysis was performed using samples of whole cell lysate or eluate from immunoprecipitation prepared in NuPage sample buffer supplemented with antioxidant reagent according to manufacturer’s specifications (Invitrogen). Samples were heated (10’ @ 70°C), then separated by electrophoresis in NuPage 4–20% polyacrylamide gels and transferred to nitrocellulose membanes using the NuPage XCell II Blow Module (Invitrogen). Blots were visualized using the Li-COR Odyssey CLX fluorescent scanner after blocking with fluorescent blocking buffer (Li-COR Biosciences), incubation with primary antibodies and fluorescently conjugated secondary antibodies (Li-COR Biosciences).
Primary antibodies: Mouse monoclonal–HNRNPC1/C2 (sc-32308, Santa Cruz), HNRNPU (3C6, Millipore), SRSF3 (7B4, Millipore), SRSF1 (AK96, Millipore); Rabbit polyclonal–FUSIP1 (A302-282A-1,Bethyl), ILF2 (A303-147A-1, Bethyl), IgG (2729, Cell Signaling), U2AF1 (A302-079A-1, Bethyl), SAM68/KHDRBS1 (sc-333, Santa Cruz), SFRS10 (AV40528, Sigma).

Whisenant T.C., Peralta E.R., Aarreberg L.D., Gao N.J., Head S.R., Ordoukhanian P., Williamson J.R, & Salomon D.R. (2015). The Activation-Induced Assembly of an RNA/Protein Interactome Centered on the Splicing Factor U2AF2 Regulates Gene Expression in Human CD4 T Cells. PLoS ONE, 10(12), e0144409.

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Western Blot Protein Detection Protocol

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For a full list of antibodies, see reporting summary. The samples were prepared in NuPAGE Sample Buffer (Invitrogen). Then proteins were separated by electrophoresis in 4–12% Bis-Tris Protein Gels and transferred to the polyvinylidene difluoride membrane (Thermo Scientific). The membranes were blocked in 5% dry milk in 0.1% Tween-20 in PBS and detected with the indicated antibodies. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (BIO-RAD), proteins were visualized using ECL detection reagents (GE Healthcare). Uncropped gel images for Western blots are available in Source Data file.

Zhang J., Bellani M.A., James R.C., Pokharel D., Zhang Y., Reynolds J.J., McNee G.S., Jackson A.P., Stewart G.S, & Seidman M.M. (2020). DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain. Nature Communications, 11, 3951.

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Cell Surface Biotinylation Assay

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We used a cell surface biotinylation assay to study protein expression at the plasma membrane. 48 h after transfection, TsA-201 cells transiently expressing Na_v1.5 constructs were supplied with 0.5 mg/mL EZ-link™ Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) in PBS for 15 min at 4°C. Then, we washed the cells twice with 200 mM glycine in cold PBS to inactivate and remove excess biotin, respectively. Cells were taken up in lysis buffer (composed of [mM] HEPES 50 pH 7.4, NaCl 150, MgCl₂ 1.5, EGTA 1 pH 8; 10% glycerol, 1% Triton X-100 (Tx100), complete protease inhibitor [Roche, Basel, Switzerland]) for 1 h at 4°C. Cell lysates were centrifuged at 16,100 rcf at 4°C for 15 min. We determined protein concentration of the supernatant with Bradford assay (Bio-Rad) [24 ], and incubated lysate equivalent to 2 mg of protein with 50 μL Streptavidin Sepharose High-Performance beads (GE Healthcare, IL, USA) for 2 h at 4°C, then washed the beads three times with lysis buffer, and eluted them with 30 μL of 2X NuPAGE Sample Buffer plus 100 mM DTT (37°C for 30 min). Input fractions were resuspended in NuPAGE Sample Buffer (Invitrogen) plus 100 mM dithiothreitol (DTT) and incubated at 37°C for 30 min.

Wang Z., Vermij S.H., Sottas V., Shestak A., Ross-Kaschitza D., Zaklyazminskaya E.V., Hudmon A., Pitt G.S., Rougier J.S, & Abriel H. (2020). Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5. Channels, 14(1), 268-286.

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Yeast Whole Cell Extract Preparation for IP Assays

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To prepare yeast whole cell extract (WCE) for IP assays, 1-liter yeast cell culture was grown in YPD medium to an O.D. of 1.5. The harvested cells were lysed and processed for WCE preparation according to a protocol described previously (23 (link),24 (link)). WCE (1.5 mg) was mixed with 75 μl of anti-FLAG antibody agarose beads (M2; Sigma-Aldrich) and incubated at 4°C for 2 h in WCE buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 5 mM MgCl₂, 1 mM EDTA and 20% glycerol). The bound proteins were washed three times with 200 μl of WCE buffer, and the proteins were extracted by boiling with NuPAGE sample buffer (Invitrogen) for subsequent SDS-PAGE and Western blot analyses. For all Western blot analyses in this study, immuno-stained protein bands were visualized using the Odyssey infrared imaging system (LI-COR Biosciences).

Shekhar A.C., Sun Y.E., Khoo S.K., Lin Y.C., Malau E.B., Chang W.H, & Chen H.T. (2022). Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation. Nucleic Acids Research, 51(9), 4223-4236.

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Western Blot Analysis of dFMRP Levels

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Studies were performed as previously described (Gatto and Broadie, 2008 (link); 2009 (link); 2011 (link)). Briefly, heads from staged animals were homogenized and boiled in NuPage sample buffer (Invitrogen, Carlsbad, CA) supplemented with 40mM DTT. Total protein from 3 heads per sample was loaded onto 4–12% Bis-Tris gels and electrophoresed in NuPage MES Buffer (Invitrogen) for 1 hour at 200 V. Transfer to nitrocellulose was performed for 1 hour at 100 V in NuPage transfer buffer (Invitrogen) with 10% MeOH. Processing was completed using the Odyssey near infrared fluorescence detection system (Li-COR, Lincoln, NE). Antibodies used included: anti-dFMRP (1:3,000; 6A15), anti-α-tubulin (1:100,000; B512, mouse, Sigma), anti-GAD (1:1,000; 819), Alexa-goat-anti-rabbit-680 (1:10,000; Invitrogen–Molecular Probes), and IR-goat-anti-mouse-800 (1:10,000; Rockland). GAD levels were quantified by measuring immunoreactive band intensities compared to the α-tubulin loading control. The dfmr1 null was normalized to w¹¹¹⁸ genetic background control to report percent expression levels.

Gatto C.L., Pereira D, & Broadie K. (2014). GABAergic circuit dysfunction in the Drosophila Fragile X syndrome model. Neurobiology of disease, 65, 142-159.

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SDS-PAGE and Western Blot Analysis

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Protein samples were denatured in NuPAGE sample buffer (Invitrogen) supplemented with 5% (v/v) 2-mercaptoethanol and by subsequent heating to 95°C for 5 min. Denatured proteins were resolved on 12% (w/v) NuPAGE polyacrylamide gels run with MOPS buffer (Invitrogen). See Blue Plus, protein molecular weight markers (Invitrogen) were run on all NuPAGE gels. Instant Blue stain (Expedeon Ltd.) was used to visualize the protein bands. Western blotting to detect either TMV coat protein with an anti-TMV antibody, (AS-72203-2ML, Austral Biologicals, San Ramon, CA, United States) or the six C-terminal histidine residues with a monoclonal antibody prepared to six histidine residues, (Cat 34660, Qiagen) was performed as described by Saunders and Lomonossoff (2015) (link).

Saunders K, & Lomonossoff G.P. (2017). In Planta Synthesis of Designer-Length Tobacco Mosaic Virus-Based Nano-Rods That Can Be Used to Fabricate Nano-Wires. Frontiers in Plant Science, 8, 1335.

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Western Blot Analysis of Protein Expression

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Adherent cells were lysed in NP-40 buffer containing protease cocktail inhibitor (Roche cat#11-836-170-001), sonicated, and centrifuged at 14,000rpm for 15 min at 4°C. Lysates were collected and protein concentration was determined using the BCA assay (Thermo cat#23225). 35–50μg of protein was prepared in NuPAGE sample buffer (Invitrogen cat#NP0007) and reducing agent (Ivitrogen cat#NP0004). Proteins were separated on a SDS-PAGE gel under reducing conditions and were transferred to nitrocellulose membranes (Bio-Rad cat#1620115). The membrane was then blocked in 1% fish gelatin blocking buffer (Amresco cat#M319) at room temperature for 1 hour and primary antibodies (1:1000 Myc-tag (71D10) rabbit monoclonal antibody, Cell Signaling Technology cat#2278; 1:1000 FLAG-tag rabbit polgyclonal antibody, Invitrogen cat#PA1-984B) were applied overnight at 4°C. GAPDH (Sigma cat#G9295) loading control was probed on membranes at 1:20,000 for 1 hour room temperature. SuperSignal West Pico Chemiluminescent Substrate (Thermo cat#34077) was used for detection.

Dang D., Prasad H, & Rao R. (2017). Secretory Pathway Ca2+-ATPases Promote In Vitro Microcalcifications in Breast Cancer Cells. Molecular carcinogenesis, 56(11), 2474-2485.

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Expression and Purification of Mouse Cadherin and Protocadherin Constructs

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Recombinant modified pcDNA3 vectors encoding the entire cytoplasmic regions of mouse cadherin-23, Pcdh15-CD2 and Pcdh15-CD3 (GenBank Accession Nos. Q99PF4, Q0ZM28, and Q0ZM20, respectively) coupled to an N-terminal Flag-tag were constructed for expression in HEK-293 cells. Cell lysates were prepared in Nupage Sample Buffer (Invitrogen).
A recombinant modified pFastbac vector encoding the cytoplasmic domain of mouse Pcdh15-CD1 (GenBank Accession No. NP_001165405) was constructed for expression in Sf9 insect cells. The N-terminally tagged fusion product, 6His-Flag-protein, was purified on a Ni-NTA column.
All samples were resolved by 4–8% Nupage SDSPAGE (Invitrogen). Proteins were transferred to PVDF membranes (Millipore) and immunoprobed, and bound antibody was detected by enhanced chemiluminescence (Pierce Biotechnology). A monoclonal anti-flag antibody was used to detect the four recombinant proteins (M2 Sigma-Aldrich, 1:500 dilution).

Pepermans E., Michel V., Goodyear R., Bonnet C., Abdi S., Dupont T., Gherbi S., Holder M., Makrelouf M., Hardelin J.P., Marlin S., Zenati A., Richardson G., Avan P., Bahloul A, & Petit C. (2014). The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells. EMBO Molecular Medicine, 6(7), 984-992.

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