Cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail. Protein extracts were resolved on a NuPAGE Bis-Tris (Thermo) gel and transferred to a PVDF membrane. Membranes were incubated overnight at 4 °C with appropriate primary antibodies. Detection of proteins was carried out by incubations with horseradish peroxidase (HRP)-conjugated secondary antibodies followed by enhanced chemiluminescence detection reagents. For serum western blots, protein from serum samples was denatured with NuPAGE (Thermo) reducing agent and then loaded onto a NuPAGE Bis-Tris (Thermo) gel. Band density was quantified using Fiji/ImageJ (NIH).
Nupage bis tris
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE Bis-Tris is a pre-cast polyacrylamide gel system designed for the separation and resolution of proteins. It utilizes a Bis-Tris buffer system and a neutral pH for effective protein separation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Nupage, by Thermo Fisher Scientific (5 mentions)
Lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (3 mentions)
Ecl western blot reagent, by GE Healthcare (3 mentions)
Horseradish peroxidase, by Agilent Technologies (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Hyperfilm, by GE Healthcare (2 mentions)
Peroxidase conjugated secondary antibody, by Cytiva (2 mentions)
Fermentas, by Thermo Fisher Scientific (2 mentions)
Pqe30xa expression vector, by Qiagen (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Western lightning, by PerkinElmer (2 mentions)
Colloidal blue staining kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
95 protocols using nupage bis tris
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Ion Channels
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Conventionally cultured Capan-1 cells were lysed in lysis buffer containing (mM) 250 TRIS, 1,25 NaCl, 20 NaF, 50 EDTA, 5% Trizol, and 1 × Sigma fast protease inhibitor (Sigma-Aldrich, Germany). The lysates were centrifuged for 15 min at 15000 g at 4 °C, and the supernatant was stored at − 80 °C until use. Protein concentration was determined in the samples by use of a Bradford assay. Ten to 20 μg of protein was loaded on 10% polyacrylamide precast gels (NuPAGE, Bis–Tris, Invitrogen), separated by electrophoresis, and transferred to a PVDF membrane (Invitrogen). Membranes were blocked in a solution with 3% BSA and 2% skim milk in TRIS-buffered saline containing 0.1% Tween (TBST) (60 to 90 min, room temperature (RT)). The membranes were incubated overnight at 4 °C with primary antibodies against KCNQ1 (1:1000 rabbit polyclonal, APC-022, Alomone Labs), TREK-2 (1:600 rabbit polyclonal, APC-055, Alomone Labs), and TASK-2 (1:2000 rabbit polyclonal, LS-C291082, (LifeSpan BioSciences, Inc.)), followed by rinsing in TBST and treatment with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000 to 1:2500 goat anti-rabbit, sc-2004 (Santa Cruz) in TBST with 3% BSA and 2% skim milk). Chemiluminescence of the immunoreactive bands was visualized with EZ-ECL (Biological Industries) on the Fusion FX (Vilber Lourmat) imaging system.
3
Western Blotting Protocol for Protein Analysis
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For western blotting, cells were washed with cold PBS, lysed in NP-40 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate) supplemented with protease inhibitors and centrifuged (20,000 g for 15 min). Protein concentration was measured using a BCA protein assay kit (Pierce) and LDS sample buffer (NuPAGE, Invitrogen) was added to the lysate or directly to the medium. Equal amounts were loaded on SDS-PAGE pre-cast gradient gels (4–12% Nu-Page Bis-Tris, Invitrogen), followed by transfer to nitrocellulose membrane. Non-specific protein binding was blocked by 5% skimmed milk in TBST followed by incubation with primary antibodies were overnight at 4°C in TBST with 2.5% skimmed milk, and then secondary antibodies conjugated to horseradish peroxidase (DAKO, Glostrup, Denmark) for 1 h at room temperature. Proteins were detected using ECL western blot reagent.
4
Immunoblotting and Immunoprecipitation of Fly Piwi Proteins
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Kc167 cell pellets and pestle-ground fly tissues were lysed by sonication in RSB-200 buffer containing 20 mM Tris–HCl pH 7.4, 200 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40, 0.1% Triton X-100, and complete EDTA-free protease inhibitors (Roche). Lysates were precleared with centrifugation at 16,000g for 20 min at 4°C. Samples were mixed with SDS-loading buffer and denatured at 70°C for 12 min. Proteins were separated by 4%–12% NuPAGE Bis–Tris and blotted to nitrocellulose membranes (Invitrogen). The following antibodies were used in this study: Aub-83 (Vourekas et al. 2016 (link)), SYM11 anti-sDMA (Millipore), E7 anti-β-Tubulin (Developmental Studies Hybridoma Bank). 4D10 anti-Aub, anti-AGO3, anti-Armi, and anti-Tudor antibodies were a gift from Dr. Mikiko Siomi. Piwi-N7 and Vasa-2 antibodies were produced by immunizing rabbits with the following synthetic peptides: Piwi (MADDQGRGRRRPLNED); Vasa-2 (KREFYIPPEPSNDA). Both were conjugated to KLH protein. Sera were affinity purified with columns containing the immobilized peptides (Genscript). Piwi-N7 successfully detected and immunoprecipitated Piwi protein and associated piRNAs in ovary, embryo, Kc167, and OSC lysates (Fig. 1 A–C; Supplemental Fig. S3A–E ). Similarly, Vasa-2 antibody detected and immunoprecipitated Vasa protein in ovary lysate (Supplemental Fig. 3F ).
5
SDS-PAGE Protein Analysis
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Samples were diluted with 4× LDS (Lithium dodecyl sulfate, Invitrogen) sample buffer, heated for 5 min at 90 °C and loaded in a final volume of 20 μL/well on SDS-PAGE gels (4–12% NuPAGE Bis–Tris, Invitrogen). Gels were run at 150–200 V for 35–50 min in 1× 2-(N-morpholino)ethanesulfonic acid (MES) sodium dodecyl sulfate (SDS) running buffer and stained with SimplyBlue™ SafeStain (Invitrogen).
6
SARS-CoV-2 S Protein and ACE2 Interaction
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A 3.5-μg aliquot of SARS-CoV-2 S protein as well as a 2-μg aliquot of human ACE2 were combined with Laemmli sample buffer, analyzed on a 4%–12% Invitrogen NuPage Bis-Tris gel using the MES pH 6.5 running buffer, and stained with Coomassie Brilliant Blue G-250.
7
Protein Expression Analysis in Cytotoxic Cells
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The samples were separated on a 4-12% NuPAGE® Bis-Tris gel in a MES buffer system (Invitrogen) and the separated proteins were transferred to PVDF membrane (Millipore). The membrane was blocked in 5% BSA buffer for 1 h and then incubated with anti-GNLY mAb (clone RF10, MBL), anti-perforin Ab (abcam), anti-granzyme B Ab (BD pharmingen), anti-cleaved caspase3, caspase7 and PARA (Cell Signaling) mAb at 4°Covernight. Proteins were detected with Immobilon™ Western chemiluminescent HRP substrate (Millipore)
8
Western Blotting of SRA1 Protein
9
Western Blot Analysis of Proteins
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Equal volumes of protein solution or amounts of total protein were used for analysis. Protein concentration was determined by the BCA protein assay (Pierce) or OD280. All samples were diluted with 1-4x SDS or LDS (Invitrogen) sample buffer, heated to 80°C for 10 min and resolved on 4–12% NuPage BisTris gradient gel in combination with MES or MOPS running buffer (Invitrogen). Western blotting was done with the respective NuPage Blot system utilizing a PVDF membrane (Millipore). The latter was blocked in PBS containing 4% nonfat milk and 0.1% Tween 20. Signal detection was carried out using HRP-conjugated secondary antibodies (Dianova), enhanced chemiluminescence (ECL) assay solution (Millipore) and hyperfilms (GE). ImageJ 1.46 (NIH) was used for signal quantification and all protein levels were normalized against those of GAPDH from the same sample.
10
Western Blot Analysis of HIF-1α and ISCU
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Cells were lysed and cytoplasmic and nuclear extracts were prepared using the NE-PER extraction kit with 1X halt-protease inhibitors cocktail (both Pierce, Waltham, MA) and EDTA. Protein extracts were stored at -80°C. 20μg of protein were mixed with warm 2X lithium dodecyl sulfate (LDS) such that the LDS to sample ratio was 1:4 (v/v). Sample were denatured for 5 minutes at 95°C and subjected to SDS-PAGE (4–12% NuPAGE Bis-Tris) with 1X MOPS running buffer diluted with dH2O (all Invitrogen). The proteins were transferred onto a nitrocellulose membrane by an iBlot apparatus for 8 minutes (all Invitrogen). The membrane was blocked with 5% w/v nonfat dry milk in 1X TBST (10nM Tris-HCl, pH 8.0, 150nM NaCl, and 0.05% Tween 20). The blot was incubated with mouse antibodies to HIF-1α (1:500, BD Biosciences, San Jose, CA) or ISCU (1:800, ab180532, Abcam) overnight and goat anti-mouse IRDye 800CW antibody (Licor Biosciences, Lincoln, NE) at a 1:10,000 dilution for 30 minutes; being washed twice with washing buffer in between incubations. Scanning was performed using the Li-Cor Odyssey CLx imaging system coupled with the Image Studio software. Images were later adjusted for contrast and intensity using PowerPoint 2011 version 14.6.3 for Macintosh (Microsoft Co, Redmond, WA).
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