Nupage lds sample buffer

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The NuPAGE LDS sample buffer is a laboratory reagent used to prepare protein samples for electrophoresis analysis. It is designed to denature and solubilize proteins prior to separation by gel electrophoresis.

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1 340 protocols using nupage lds sample buffer

Chromatin Fractionation and Analysis

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4x10⁶ eHAP cells were seeded per 10cm dish 24 h prior to collection. Cells were treated with 10uM formyl-dU or DMSO control for 1 h prior to collection. Cells were scraped in 1ml ice-cold PBS. 50% of the sample was kept on ice for whole cell control. The remaining cells were spun down for 4 min at 500 g and resuspended in 200ul CSK buffer (10mM PIPES pH7.0, 100mM NaCl, 300mM Sucrose, 1.5mM MgCL₂, 5mM EDTA, 0.5% Triton 1x phosphatase (Phos-Stop, Roche) and protease (Complete, EDTA-free, Roche) inhibitor mixes)) and incubated on ice for 10 min. Cells were spun down at full speed for 10 s 150ul of supernatant (soluble fraction) was collected. Residual soluble fraction was removed and the chromatin pellet was washed in 500ul of CSK. Whole cell chromatin pellets were resuspended in 200ul 1X NuPAGE LDS sample buffer (Invitrogen) and 1% 2-metcaptoethanol (Sigma-Aldrich). 50uL of 4x NuPAGE LDS sample buffer (Invitrogen) and 4% 2-metcaptoethanol (Sigma-Aldrich) was added to soluble fraction. Samples were sonicated with a probe at medium intensity for 10 s in a Soniprep 150 instrument and then incubated at 95°C for 10 min. 20ul of each fraction was loaded and subjected to SDS-PAGE as above.

Hewitt G., Borel V., Segura-Bayona S., Takaki T., Ruis P., Bellelli R., Lehmann L.C., Sommerova L., Vancevska A., Tomas-Loba A., Zhu K., Cooper C., Fugger K., Patel H., Goldstone R., Schneider-Luftman D., Herbert E., Stamp G., Brough R., Pettitt S., Lord C.J., West S.C., Ahel I., Ahel D., Chapman J.R., Deindl S, & Boulton S.J. (2021). Defective ALC1 nucleosome remodeling confers PARPi sensitization and synthetic lethality with HRD. Molecular Cell, 81(4), 767-783.e11.

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Intrabody Expression Analysis by Western Blot

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Medium collected from the SH-SY5Y cells transfected with mRNA encoding the IgG HC and the IgG LC and co-transfected with both was diluted in 4× NuPage LDS Sample Buffer (Thermo Fisher Scientific). For reduced conditions, 10× NuPage Reducing Agent (Thermo Fisher Scientific) was added and samples were heated at 70°C for 10 min. Samples were electrophoresed and western blotted with the anti-mouse secondary antibody only. To determine the expression of the RNJ1 intrabody, SH-SY5Y cells transfected with the mRNA encoding the RNJ1 scFv were lysed in 1× RIPA buffer (Abcam), and total protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific). Three- to five-microgram total protein was diluted in 4× NuPage LDS Sample Buffer (Thermo Fisher Scientific) with 10× NuPage Reducing Agent (Thermo Fisher Scientific) and heated at 70°C for 10 min. Samples were electrophoresed and western blotted with anti-Flag antibody (Cell Signalling Technology). All membranes were imaged using the Odyssey Fc Imaging System (LI-COR). Image processing was performed on Image Studios software (LI-COR).

Wongsodirdjo P., Caruso A.C., Yong A.K., Lester M.A., Vella L.J., Hung Y.H, & Nisbet R.M. (2024). Messenger RNA-encoded antibody approach for targeting extracellular and intracellular tau. Brain Communications, 6(2), fcae100.

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Cellular Fractionation and Protein Extraction

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Cells were lysed and resuspended in NP40 lysis buffer: 0.5% NP40, 10 mM Tris HCl pH 7.8, 10 mM EDTA, 150 mM NaCl, 2.5 mM Na orthovanadate, 1 × protease inhibitor cocktail (Roche 11836170001). The lysate was sonicated in an ultrasonic bath and centrifuged at 21000xg for 25 min. The supernatant (soluble fraction) was collected and boiled in 1X NuPage™ LDS-Sample buffer (Invitrogen, NP0007) at 95 °C for 5 min. The pellet was washed by resuspending in Washing buffer: 50 mM Tris HCl pH 7.4, 150 mM NaCl, and centrifuged at 21000xg for 5 min. The pellet was then resuspended in resolubilization buffer: 50 mM Tris HCl pH 6.8, 5% SDS, 10% glycerol followed by sonication and centrifugation at 12000xg for 10 min. The supernatant (insoluble fraction) was collected and boiled in 1X NuPage™ LDS-Sample buffer (Invitrogen, NP0007) at 95 °C for 5 min.

Fortuna T.R., Kour S., Anderson E.N., Ward C., Rajasundaram D., Donnelly C.J., Hermann A., Wyne H., Shewmaker F, & Pandey U.B. (2021). DDX17 is involved in DNA damage repair and modifies FUS toxicity in an RGG-domain dependent manner. Acta neuropathologica, 142(3), 515-536.

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2D Proteomic Analysis of Rickettsial Proteins

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Rickettsial protein extracts were prepared from purified bacteria as previously described (Pornwiroon et al., 2009 (link)), and 30 μg of protein were subjected to isoelectric focusing (IEF) in a immobilized pH gradient strip (7 cm, linear pH 3 to 10; Bio-Rad, Hercules, CA) using a PROTEAN IEF cell apparatus (Bio-Rad) at 20 °C as described previously (Pornwiroon et al., 2009 (link)). The IEF strip was sequentially equilibrated for 15 min each in 1X NuPAGE LDS sample buffer (Invitrogen) containing 1% (w/v) dithiothreitol and 1X NuPAGE LDS sample buffer containing 2.5% (w/v) iodoacetamide. After the equilibration, the strip was placed on top of NuPAGE Novex 4 to 12% Bis-Tris ZOOM gel (Invitrogen) and sealed with 0.5% agarose. Electrophoresis was performed in the XCell SureLock Mini-Cell System (Invitrogen) at 100 V until the tracking dye reached the gel bottom. Gels were fixed in fixative solution (10% methanol/7% acetic acid) for 1 h and overnight stained with SYPRO Ruby protein gel stain (Bio-Rad). Gels were digitized using Molecular Imager Gel Doc XR System (Bio-Rad) and stored at 4 °C for spot excision and protein identification.

Pornwiroon W., Bourchookarn A., Paddock C.D, & Macaluso K.R. (2015). Immunoproteomic profiling of Rickettsia parkeri and Rickettsia amblyommii. Ticks and tick-borne diseases, 6(6), 829-835.

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Protein Extraction and Western Blot Analysis

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Protein extraction and western blot experiments on whole testes and elutriated spermatids were performed as described in Comptour et al.⁶⁵ (link) In brief, 15 μl of each immunoprecipitated sample and a volume of input sample corresponding to 10% of IP sample were denatured using 4 × NuPAGE LDS sample buffer (Life Technologies) with 10% β – mercaptoethanol, boiled for 10 min at 95 °C and loaded. Extraction of sperm proteins was as follow: five millions of spermatozoa (purity ⩾99%) were resuspended in 200 μl of 4 × NuPAGE LDS sample buffer (Life Technologies) and boiled for 10 min at 95 °C. Ten microliter of sample were loaded per lane. Antibody against SLY^{14 (link)} was diluted 1/3000, antibody against SLX/SLXL1,^{69 (link)} 1/6000, anti-H3K79me2 (ab-3594 from Abcam), anti-TBLX1R1 (ab 24550 from Abcam), anti-Tubulin antibody (T-9026 from Sigma-Aldrich), anti-panH3 antibody (05-928 from Abcam), anti-FLAG (M5 from Sigma-Aldrich) and anti-Hup2B antibody (Briar Patch Biosciences, Grass Valley, CA, USA), 1/1000.

Moretti C., Serrentino M.E., Ialy-Radio C., Delessard M., Soboleva T.A., Tores F., Leduc M., Nitschké P., Drevet J.R., Tremethick D.J., Vaiman D., Kocer A, & Cocquet J. (2017). SLY regulates genes involved in chromatin remodeling and interacts with TBL1XR1 during sperm differentiation. Cell Death and Differentiation, 24(6), 1029-1044.

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SDS-PAGE Protein Extraction and Western Blot

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Cell pellets were lysed in RIPA buffer (Sigma-Aldrich), mixed with 4x NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) + 10% dTT and incubated at 95°C for 15 min. If necessary, samples were sonicated prior to adding the LDS sample buffer. In vitro reactions were mixed with 4x NuPAGE LDS Sample Buffer (Thermo Fisher Scientific) + 10% dTT and incubated at 95°C for 15 min. Samples were loaded onto a SDS-PAGE Gel (Invitrogen) and run in 1x MOPS SDS Running Buffer (Invitrogen). The protein was transferred onto a nitrocellulose membrane using the iBlot1 system (Invitrogen) which was then blocked in 5% milk (Marvel) diluted in PBS+0.01% Tween (PBST). The primary and secondary antibodies were also diluted in 5% milk in PBST and the membrane was washed three times with PBST after antibody incubations. HRP-coupled secondary antibody was detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Bottermann M., Foss S., Caddy S.L., Clift D., van Tienen L.M., Vaysburd M., Cruickshank J., O’Connell K., Clark J., Mayes K., Higginson K., Lode H.E., McAdam M.B., Sandlie I., Andersen J.T, & James L.C. (2019). Complement C4 Prevents Viral Infection through Capsid Inactivation. Cell Host & Microbe, 25(4), 617-629.e7.

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Western Blot Analysis of Respiratory Syncytial Virus

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Infected Vero cells from single-cycle infections were harvested in 1X NuPage LDS sample buffer (Thermofisher) diluted in PBS containing protease inhibitor (Roche). Cell lysates were homogenized using a QIAshredder spin column (Qiagen) and protein concentrations were determined by BCA assay (Pierce BCA Protein Assay kit). Thirty μg of proteins was denatured in a final composition of 1X NuPage LDS sample buffer (ThermoFisher) and 1X NuPAGE Sample Reducing Agent (Invitrogen) by heating at 90°C for 10 min before being subjected to electrophoresis on NuPAGE 4–15% Mini-PROTEAN TGX Gels (Bio-Rad) with 1X TGS Running Buffer (Bio-Rad). Odyssey Protein Molecular Weight Marker (Li-Cor) was run in parallel. Proteins were transferred to PVDF membranes using the iBlot 2 Gel Transfer Device (ThermoFisher) and stained with a primary mouse anti-RSV F (abcam, ab43812), a mouse anti-RSV P (abcam, ab94965) or a mouse anti-RSV G (abcam, 94966) antibody. The rabbit anti-Tubulin (abcam, ab52866n) antibody was used as a loading control. The secondary antibodies used were goat anti-rabbit IgG IRDye 680RD (at 1:15,000, Li-Cor, 926–68071), and goat anti-mouse IgG IRDye 800CW (1:10,000, Li-Cor, 926–32210). Membranes were scanned using Odyssey software, version 3.0 (Li-Cor).

Levy M., Chen J.W., Kaiser J.A., Park H.S., Liu X., Yang L., Santos C., Buchholz U.J, & Le Nouën C. (2024). Intranasal respiratory syncytial virus vaccine attenuated by codon-pair deoptimization of seven open reading frames is genetically stable and elicits mucosal and systemic immunity and protection against challenge virus replication in hamsters. PLOS Pathogens, 20(5), e1012198.

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Western Blot Analysis of MARV and RAVV GP

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One microgram aliquots of purified MARV or RAVV GP protein constructs were equilibrated with NuPAGE LDS Sample Buffer (ThermoFisher Scientific) for non-reducing conditions, or equilibrated with NuPAGE LDS Sample Buffer, reduced by addition of DTT up to a final concentration of 50 mM, and heated at 95°C for 5 min for reducing conditions. The samples were resolved on SDS-PAGE gels and transferred on to polyvinylidene difluoride membranes (MilliporeSigma). The membranes were blocked with 5% milk and 0.05% Tween-20 in PBS for 1 h at ambient temperature and probed overnight at 4°C with MR228 or MR235 antibodies diluted to 1 μg/mL in 3% milk and 0.05% Tween-20 in PBS. Next, the membranes were washed 3 times in PBS with 0.05% Tween-20, and incubated with goat anti-human IgG Fc-HRP secondary antibody (Invitrogen) diluted at 1:5,000 in 3% milk and 0.05% Tween-20 in PBS for 1 h at ambient temperature. The protein bands were visualized using ECL substrate (ThermoFisher Scientific).

Ilinykh P.A., Huang K., Santos R.I., Gilchuk P., Gunn B.M., Karim M.M., Liang J., Fouch M.E., Davidson E., Parekh D.V., Kimble J.B., Pietzsch C.A., Meyer M., Kuzmina N.A., Zeitlin L., Saphire E.O., Alter G., Crowe JE J.r, & Bukreyev A. (2020). NON-NEUTRALIZING ANTIBODIES FROM A MARBURG INFECTION SURVIVOR MEDIATE PROTECTION BY FC-EFFECTOR FUNCTIONS AND ENHANCING EFFICACY OF OTHER ANTIBODIES. Cell host & microbe, 27(6), 976-991.e11.

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Western Blot Analysis of AGR2 and AGR3

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Each 100ng of recombinant human AGR2 and AGR3 protein (Biorbyt, Cambridge, UK) was diluted 1:2 in 2x NuPAGE LDS Sample Buffer (Invitrogen, Carlsbad, CA, USA) supplemented with 5% dithiothreitol and heat denatured (5min, 95°C). For separation protein was loaded on 4–12% gradient gels (NuPAGE; Invitrogen) and then transferred onto 0.2μm PVDF membranes (Whatman, Dassel, Germany) (1h, 100V) for immunodetection. Blots were blocked in TRIS-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% non-fat dry milk (Merck, Darmstadt, Germany) overnight at 4°C. Blocked blots were probed with mouse monoclonal anti-AGR3-antibody (ab82400, Abcam, Cambridge, UK), diluted 1:500 in blocking solution, for 1h at room temperature. After washing (TBS + 0.05% Tween-20), blots were incubated with rabbit anti-mouse (DAKO, Glostrup, Denmark) secondary peroxidase-conjugated antibody, diluted 1:4000 in blocking solution, for 1h at room temperature. After washing (TBS + 0.05% Tween-20), antibody detection was accomplished with Pierce ECL Western blotting Substrate (Thermo Scientific, Rockford, USA). Original uncropped blot is depicted in the supplements (S1 Fig.).

Garczyk S., von Stillfried S., Antonopoulos W., Hartmann A., Schrauder M.G., Fasching P.A., Anzeneder T., Tannapfel A., Ergönenc Y., Knüchel R., Rose M, & Dahl E. (2015). AGR3 in Breast Cancer: Prognostic Impact and Suitable Serum-Based Biomarker for Early Cancer Detection. PLoS ONE, 10(4), e0122106.

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Immunoblot Analysis of CHD1 and CHD2

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The antibodies used for immunoblot analysis were CHD1 (Bethyl Laboratories), CHD2 (Cell Signaling), α/β-Tubulin (Cell Signaling) and Alexa Fluor 680 donkey anti-rabbit IgG (Invitrogen). 48 h post-transfection cells were lysed in 2x NuPAGE LDS sample buffer (Invitrogen) and resolved by SDS-PAGE on NuPAGE 4-12% Bis-Tris gels (Invitrogen). Proteins were transferred to Immobilon-FL PVDF membranes (Millipore) and membranes were for blocked for 1 h in 0.1% Casein/0.2x PBS (Biorad). The membrane was then probed with primary antibody in blocking buffer +0.1% Tween-20 overnight at 4°C. Membranes were washed three times with PBS +0.1% Tween-20, then incubated with Alexa Fluor 680 donkey anti-rabbit secondary antibody in blocking buffer +0.1% Tween-20 + 0.01% SDS for 1 h at room temperature. Immunoblots were washed, then visualized and quantitated using an Odyssey infrared imaging system (Licor). CHD1 and CHD2 protein levels were normalized to α/β-Tubulin levels.

Rodgers M.J., Banks D.J., Bradley K.A, & Young J.A. (2014). CHD1 and CHD2 are positive regulators of HIV-1 gene expression. Virology Journal, 11, 180.

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