SVD-VLP, mock-infected Sf9 cell lysates, and SVDV grown in ST cells prepared in the same manner as SVD-VLP were separated with 10% NuPAGE Novex Bis-Tris gels (Invitrogen) followed by protein transfer to nitrocellulose membranes using a iBlot gel transfer device (Invitrogen). Membranes were incubated in PBS-T with 5% skim milk at room temperature for 1 h then blotted with anti-mouse SVD serum (1:500) in blocking buffer overnight at 4℃, followed by incubation with HRP-conjugated anti-mouse antibody (1:2,000; Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Blots were developed by using 3, 3′-diaminobenzidine (Sigma-Aldrich).
Nupage novex bis tris gel
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
NuPAGE Novex Bis-Tris gels are precast polyacrylamide gel electrophoresis (PAGE) products designed for the separation and analysis of proteins. These gels utilize a Bis-Tris buffer system for improved resolution and protein separation.
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Lab products found in correlation
Pvdf membrane, by Merck Group (10 mentions)
Hybond, by Cytiva (8 mentions)
Whatman, by Cytiva (6 mentions)
Dc protein assay, by Bio-Rad (6 mentions)
Iblot gel transfer device, by Thermo Fisher Scientific (5 mentions)
Odyssey infrared imaging system, by LI COR (5 mentions)
Complete protease inhibitor, by Roche (5 mentions)
Las 4000, by Cytiva (5 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (5 mentions)
Trypsin, by Promega (4 mentions)
Anti gapdh 6c5, by Merck Group (4 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
3 3 diaminobenzidine (dab), by Merck Group (3 mentions)
Iblot system, by Thermo Fisher Scientific (3 mentions)
Ecl detection system, by GE Healthcare (3 mentions)
Non fat dry milk, by Bio-Rad (3 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Qproteome cell compartment kit, by Qiagen (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
161 protocols using nupage novex bis tris gel
1
Western Blot Analysis of SVD Antigens
2
Western Blot Protein Separation and Detection
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Cells were washed twice with PBS, and lysed with 95 °C SDS-sample buffer containing 4% β-mecapto-ethanol. Samples were boiled at 95 °C for 4 min to denature proteins. Proteins were separated on 4–12% NuPAGE Novex Bis-Tris gels (Invitrogen), transferred to Immobilon-PVDF transfer membranes (Millipore Corp., Billerica, MA) and subsequently blocked with 2.5% (w/v) BSA in Tris-buffered saline with Tween 20 for 60 min. The immunoblots were analysed using primary antibodies incubated overnight at 4 °C and secondary antibodies linked to horseradish peroxidase (HRP) (GE Healthcare, UK), after each step immunoblots were washed 6 × with Tris-buffered saline with Tween 20. Signals were visualized by enhanced chemiluminescence and light sensitive films (GE Healthcare, UK). The full blots of all immunoblots presented in the study are shown in Supplementary Fig. 8 .
3
Protein Extraction and Quantification
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For total protein extracts, 8 M urea lysis buffer was used (8 M urea, 0.5% [v:v] Triton X-100 [Sigma-Aldrich, X100], DTT [100 mM], Complete® protease inhibitor [PI; Roche, 11873580001]), long with phosphatase inhibitor cocktail (PIC) 2 and 3 (Sigma, P5726 and P0044). Protein concentration was determined by Bio-Rad protein assay (Bio-Rad 500–0006). Proteins (50 µg) were separated using NuPAGE® Novex® Bis-Tris Gels (Invitrogen, NP0322, NP0321, WG1403, WG1402A), transferred to nitrocellulose membranes using Invitrogen's iBlot system. Bound antibodies were imaged using appropriate near-infrared (NIR) dye conjugates (Li-Cor) and an Odyssey NIR scanner (Li-Cor Biosciences) or using HRP-linked secondary antibodies (Dako) and SuperSignal West Femto substrate (Pierce, 34096) with a Li-Cor Odyssey Fc System. Image studio 3.1 was used for quantification. For statistical assessment, mean intensity values were used, while bar graphs of quantifications show fold changes.
4
Protein Fractionation and Sequencing
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The total proteins extracted from HEp-2 cells were fractionated in a DS-Sepharose column with a BioLogic Duo-Flow system (Bio-Rad). About 40 mg of proteins in 40 mL of 10 mM phosphate buffer (pH 7.4; buffer A) were loaded into the DS-Sepharose column at a rate of 1 mL/min. After loading, the column was washed with 60 mL of buffer A to remove excess and non-binding proteins, followed by eluting with 40 mL of 0.2 M NaCl in buffer A to further remove very weakly binding proteins. Proteins with low to high DS-affinity were eluted with sequential 40-mL step gradients of 0.4 M, 0.6 M, and 1.0 M NaCl in buffer A. Fractions were desalted and concentrated to 0.5 mL with 5-kDa cut-off Vivaspin 20 centrifugal filters (Sartorius). The protein concentration of each fraction was measured. Fractionated proteins were further separated by 1-D SDS PAGE using 4–12% NuPAGE Novex Bis–Tris gels with MES running buffer (Invitrogen). Each gel lane was divided into three sections and subjected to protein sequencing.
5
Proteomic Analysis of GAL4-PCGFs
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Proteins from GAL4-PCGFs purification were separated by SDS–PAGE, using 4%–12% NuPAGE Novex Bis–Tris gels (Invitrogen) and NuPAGE MES SDS running buffer (Invitrogen) and then stained with Coomassie Blue using InstantBlue Comassie (Expedeon). Bands from gel were cut and digested with trypsin (Promega) and incubated for 16 h at 37°C for protein digestion. Then, peptide extraction was carried out and the resulting peptides mixture were combined, reduced in volume in a vacuum concentrator, desalted and concentrated using StageTip (Proxeon Biosystems) columns, washed with 30 μL of 0,1% Formic acid (FA) and finally eluted with 40 μL of 80% MeCN in 0,1% FA. The samples were concentrated in vacuum concentrator (Eppendorf concentrator 5301) for 5 min and peptides were dissolved in 7 μL of 0,1% FA. Approximately 5 μL of purified peptide mixture were analyzed on a LC–ESI–MS-MS Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific). Full scan MS spectra were acquired in a range of m/z 300–1800.
6
Saliva Protein Profiling by SDS-PAGE
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Sodium dodecyl sulfate PAGE (SDS-PAGE) was carried out on saliva samples. Saliva samples were prepared for electrophoresis by dilution 4 × concentration LDS sample buffer (Invitrogen) with the addition of 0.5M DTT (Sigma) to the sample-buffer solution and then boiled for 3 min. Pre-cast 4–12% NuPAGE Novex Bis-Tris gels (Invitrogen) were assembled in a XCell vertical electrophoresis unit (Invitrogen) with MES running buffer (Invitrogen). Samples were loaded with equal protein concentration and electrophoresed for 32 min at 125 mA and 200 V (constant). Molecular masses were determined by comparison with SeeBlue Plus2 standard proteins (Thermo Scientific).
7
Western Blot for WNT7b and CDK6
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Cell pellets were lysed in RIPA buffer and the lysates were cleared by centrifugation at 20,800×g for 10 min. Proteins were separated on 4–12% NuPAGE Novex Bis-Tris gels (Invitrogen), transferred to nitrocellulose (Schleicher and Schuel) or Immobilon (Millipore) membrane and probed with antibodies. Immuno-complexes were visualized by enhanced chemo-illuminescence (Pierce 34080) using goat anti-mouse (AMI3404, Biosource) or goat anti-rabbit (ALI0404, Biosource) antibodies coupled to horseradish peroxidase. The primary antibodies used were: WNT7b: Abcam (ab94915); CDK6: Thermo (AHZ0232).
8
Western Blot Analysis of Esrrβ Protein
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For western blot analysis, samples were loaded on 4–12% NuPAGE Novex Bis-Tris gels (Invitrogen, Carlsbad, CA), separated, and blotted onto polyvinylidene fluoride (PVDF) membrane (Westran S, Whatman Inc., Sanford, ME). For immunodetection, membranes were blocked in 5% skimmed milk powder, and anti-Esrrβ polyclonal antibody (1:500; Abcam, Cambridge, MA; ab19331) was applied overnight at 4 °C. The horseradish-peroxidase-coupled secondary antibodies (goat anti-rabbit-HRP 1:5000; Sigma-Aldrich, St. Louis, MO; A0545) were visualized using an enhanced chemiluminescence (ECL) detection system (GE Healthcare Amersham, Freiburg, Germany). To ensure that immunoreactivity was derived from equal protein amounts of homogenates, staining with rabbit anti-β-actin polyclonal antibody (1:300; Sigma-Aldrich; A2066) was conducted. Densitometric measurement was performed using the ImageJ 1.46o software (National Institutes of Health, Bethesda, MD).
9
Kidney Protein Fractionation and Quantification
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Kidney protein-extracts (20 μg aliquots) were fractionated by SDS-PAGE on 4–12% NuPAGE® Novex® Bis-Tris gels (Invitrogen, Grand Island, NY). Separated proteins were transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) at 300 mA for 90 min at room temperature. Membranes were blocked in TBS-T (20 mM Tris pH 7.5, 500 mM NaCl, 0.1% Tween 20) supplemented with 5% non-fat dry milk (Bio-Rad) for 1 h at room temperature. Blots were probed with the following primary antibodies: goat anti-Aminopeptidase N (AP-N), goat anti-Meprin A subunit alpha (Mep-1) and rabbit anti-glutamyl aminopeptidase (AP-A) (R&D system, 1:1000), rabbit anti-fatty acid binding protein heart type (FABPH) (1:200, Gene Tax), rabbit anti-11-fold increase in NADH dehydrogenase [ubiquinone] 1 beta sub complex subunit 9 (CI-B22) (1:500, Gene Tax) and mouse anti-vinculin (1:2000, Cell Signaling) with the corresponding horseradish peroxidase-conjugated secondary antibody (1:5000–1:20,000 dilution). Blots were processed using an ECL detection kit (GE Life, Pittsburgh, PA) and exposed to X-ray films. For quantification, films were scanned using a Bio-Rad GS-800 calibrated densitometer running Quantity One software (Bio-Rad). Optical density of each specific band was analyzed using Image J Software.
10
Quantitative Insulin Immunoblotting
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Proteins (3 mg/lane by BCA protein assay) were separated on 4–12% NuPAGE Novex Bis-Tris gels (Invitrogen), electrotransferred to nitrocellulose (Bio-Rad), and immunoblotted with guinea pig anti-insulin (Linco/Millipore) and mouse anti-tubulin (Sigma). Horseradish peroxidase-conjugated secondary antibodies were from Jackson ImmunoResearch with proteins visualized by enhanced chemiluminescence (ECL, Millipore).
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