Soluble MP-GFP expression, IB formation, and basal expression levels were analyzed with WBs according to Fink et al. [86 (link)]. For extraction NuPAGE® Sample Reducing Agent (10X) (Novex) was additionally added to a concentration of 4 mM to the lysis buffer, respectively. MP-GFP proteins was captured with Anti-GFP monoclonal antibody (Sigma-Aldrich, G6539) and detected with alkaline phosphatase‐labeled anti‐mouse IgG (whole molecule) (A5153; Sigma‐Aldrich).
Nupage sample reducing agent 10x
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NuPAGE Sample Reducing Agent (10X) is a concentrated solution used to reduce disulfide bonds in protein samples prior to electrophoresis. It is designed for use with the NuPAGE electrophoresis system.
Automatically generated - may contain errors
Lab products found in correlation
Nupage lds sample buffer 4x, by Thermo Fisher Scientific (13 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (3 mentions)
Nupage 4 12 bis tris, by Thermo Fisher Scientific (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Nupage mops sds running buffer, by Thermo Fisher Scientific (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin heat shock fraction, by Merck Group (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Invitrolon polyvinylidene difluoride membranes, by Thermo Fisher Scientific (2 mentions)
Rat igg2a anti mouse mcpt8 antibody clone tug8, by BioLegend (2 mentions)
Bis tris gels with mops running buffer, by GenScript (2 mentions)
Mouse igg1 anti gapdh antibody clone ga1r, by Cell Sciences (2 mentions)
M per, by Thermo Fisher Scientific (2 mentions)
Anti gfp monoclonal antibody, by Merck Group (1 mentions)
Egfr 4267, by Cell Signaling Technology (1 mentions)
Pak1 2602, by Cell Signaling Technology (1 mentions)
Fak 3285, by Cell Signaling Technology (1 mentions)
Trans blot semi dry electrophoretic transfer cell, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
22 protocols using nupage sample reducing agent 10x
1
Quantification of Soluble MP-GFP Expression
2
Protein Extraction and Electrophoresis
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Cell disintegration and protein extraction was performed according to Fink et al. [86 (link)]. For extraction NuPAGE® Sample Reducing Agent (10X) (Novex) was additionally added to a concentration of 4 mM to the lysis buffer, respectively. Proteins of interest were detected on SDS–polyacrylamide electrophoresis (PAGE) gel (Invitrogen NuPAGE® 4–12% Bis–Tris) according to Stargardt et al. [18 (link)].
3
Western Blot Analysis of Cell Signaling
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Total cell lysates were prepared in RIPA Buffer (10 mM NaPO4, pH 7.2, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Na-deoxycholate, 1% Nonidet P40) containing Complete EDTA-free Protease Inhibitor Cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche). Lysates were solubilized in NuPAGE LDS Sample Buffer (4X) (Life Technologies) with NuPAGE Sample Reducing Agent (10X) (Life Technologies) added to 1X. Lysates were separated by electrophoresis with 20 µg of protein per lane in a NuPAGE 4–12% Bis-Tris Gel (Life Technologies) and transferred to an Immobilon-FL 0.45 µm Pore Size Transfer Membrane (Millipore) using a Trans-Blot Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). Membranes were blocked and all antibody dilutions were performed in 5% BSA (Sigma) in TBST (10 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). All washes were performed in TBST. Primary antibodies included α-tubulin #05829 (Upstate Biotechnology), HER2 #MS-325 (Neomarkers), EGFR #4267 (Cell Signaling Technology), PAK1 #2602 (Cell Signaling Technology), Src #2110 (Cell Signaling Technology), Phospho-Src Y416 #6943 (Cell Signaling Technology), FAK #3285 (Cell Signaling Technology), and FAK Y576/577 #3281 (Cell Signaling Technology). Secondary antibodies were Alexa Fluor Conjugated Affinity Purified Anti-Rabbit or Anti-Mouse IgG (Life Technologies) detected using an Odyssey Infrared Imaging System (LiCor Biosciences).
4
Western blot analysis of HA-tagged proteins
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For Western blot analysis, 1×106 cells were lysed with 150 µL lysis buffer (50 mM Tris-HCl [pH 8], 0,1% Tween-20, 0.1% SDS, 150 mM NaCl, 0.5 mM EDTA, 10 mM DTT, and 1 mM PMSF, plus protease inhibitor cocktail; Sigma, Oakville, ON, Canada) and incubated for 20 minutes on ice. NuPage LPS loading buffer (4x) and NuPage Sample Reducing Agent (10x) (Life Technologies, Burlington, ON, Canada) were added and samples were heated for 15 minutes at 95°C. Lysates were loaded onto 4%-12% NuPage Novex BIS-Tris SDS-polyacrylamide gels (Life Technologies, Burlington, ON, Canada) and electroblotted in MOPS transfer buffer to nitrocellulose membrane (Life Technologies, Burlington, ON, Canada). Rabbit polyclonal anti-HA (Abcam, Cambridge, England) or mouse monoclonal anti-beta-actin (abm, Richmond, BC, Canada) and Mouse TrueBlot ULTRA HRP-conjugated anti-mouse (Rockland Inc., Gilbertsville, PA, USA) or goat anti-rabbit IgG antibodies (Jackson ImmunoResearch Laboratories Inc., PA, USA) in 1∶5000 dilutions of 0.1% Tween-20, 5% bovine serum albumin (BSA), Tris-buffered saline (TBS) were used for protein detection. Proteins were visualised using Clarity Western enhanced chemiluminescence (ECL) Substrate (Bio-Rad, Hercules, CA, USA).
5
Purification and Analysis of NSP10 Variants
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10 kDa centrifuge filters were purchased from Sigma-Aldrich. Five ml HisTrap FF crude columns were bought from GE Healthcare. NuPAGE MES SDS Running Buffer (20 X), SeeBlue Plus2 Pre-stained Protein Standard, SimpleBlueTM SafeStain, NuPAGE Sample Reducing Agent (10 X), NuPAGE LDS Sample Buffer (4 X) and NuPAGENovex 4–12% Bis-Tris Protein Gels (1.0 mm 10 well) were obtained from Life Technologies. Quick StartTM Bradford 1 x Dye Reagent was from Bio-Raid. SnakeSkin Dialysis Tubing was purchased from Thermo Scientific. Linbro plates were bought from Hampton Research. NT. 115 premium capillaries were obtained from Nanotemper. NSP10 expression clones for mutants T12I, T102I, and A104V optimized for E. coli expression was purchased from Genscript.
6
SDS-PAGE Protein Analysis of R36A
7
Affinity Purification of Protein Complexes
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Cells were lysed in lysis buffer [30 mM Tris pH 7.4, 150 mM NaCl, 0.5 % Nonidet P40 (v/v), 1 % protease inhibitor cocktail (v/v), 1 % phosphatase inhibitor cocktails 1 and 2 (v/v) in dH2O]. For immunoprecipitation, lysates were incubated with 20-50 μl antibody pre-bound to GammaBind G Sepharose beads (GE Healthcare, 17-0885-01) for 2 hours at 4°C, washed and resuspended in 25 μl 2x SDS-PAGE sample buffer [50 % NuPAGE LDS sample buffer 4X (Life Technologies, NP0008) (v/v), 20 % NuPAGE sample reducing agent 10X (Life Technologies, NP0004) (v/v) in dH2O] for protein gel analysis. The SF-TAP was performed as previously described by Gloeckner et al.25 (link) with minor modifications as outlined in Fig. 1A and in our recent manuscript.18 (link)
8
Protein Extraction and Western Blot Analysis
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Protein extraction was performed after 48 hours of LA treatment using a RIPA Lysis and Extraction Buffer (Life Technologies, Marly-le-Roi, France). Protein concentration was determined with a Pierce bovine serum albumine (BSA) Protein Assay kit (Thermo Scientific) by absorbance measurement at 562 nm. NuPAGE LDS Sample Buffer (4X) and NuPAGE Sample Reducing Agent (10X) (Life Technologies) were mixed with 30 μg of protein; H 2 O was added for a final volume of 15 μL. The mixture was incubated for 10 minutes at 70°C. After gel migration for 45 minutes at 200 V (NuPAGE Novex 4%-12% and 20X NuPAGE MOPS SDS Running Buffer, Life Technologies), proteins were transferred into a membrane using a Blot Dry Blotting System (Invitrogen, Paris, France). Nonspecific sites were saturated with an ECL Advance Blocking Agent (GE Healthcare, Velizy, France). Primary and secondary antibodies were diluted (1/10,000) in BSA 3% and tris buffered saline (TBS) 1%. Antibodies were incubated for at least 1 hour (APC Antibody [C-20], sc-896 Santa Cruz Biotechnology; antirabbit Antibody, DAKKO; Cyclin antibody sampler Kit #9869; Cell Signal Technology, Saint Quentin-en-Yvelines, France). The detection was performed with an ECL Advance Western Blotting Detection Kit (GE Healthcare). Quantification was achieved by densitometry analysis.
9
Quantification of Viral Envelope Protein E2
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The cell lysates prepared in DDM were analyzed for E2 content. NuPAGE LDS sample buffer (4x) (Thermo Fisher Scientific) and NuPAGE Sample Reducing Agent (10X) (Thermo Fisher Scientific) were added to the samples and heated for 10 min at 70°C for denaturing electrophoresis. The samples together with a protein molecular marker (Bio-Rad, Cat No. 1610376) were loaded on a NuPAGE Novex 10% Bis-Tris gel (Invitrogen) and run in an XCell II SureLock Mini-Cell (Invitrogen). The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using electroblotting with 1x NuPAGE transfer buffer (Invitrogen) + 10% ethanol. Membranes were blocked with BSK and stained with anti-E2 primary mAb, AP33 [55 (link)] and anti-beta actin antibody (ab8227, abcam) at 4°C overnight. The protein bands were visualized with Alexa Flour 488 goat anti-mouse IgG (Invitrogen) and Alexa Flour 647 goat anti-rabbit IgG (Invitrogen) for 1 hour at room temperature and detected using Image Lab 5.2.1, Bio-Rad.
10
Cell Surface Protein Biotinylation Assay
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For cell-surface biotinylation assay, Pierce Cell Surface Protein Isolation Kit (89881, Thermo Fisher Scientific) was used according to manufacturer’s instruction with modifications. Briefly, the cells were incubated twice for 30 min at 4 °C with biotin solution, followed by quenching. The cells were lysed with RIPA buffer (Cell Signalling Technology), and the lysates adjusted for protein concentration. Following incubation with NeutrAvidin beads, the proteins were eluted by boiling 5 min at 95 °C with NuPAGE LDS Sample buffer 4X (NP0007, Thermo Fisher Scientific) and NuPAGE Sample Reducing Agent 10X (NP0009, Thermo Fisher Scientific).
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