To the RIPA exosomes lysates an appropriate amount of 4x loading buffer was added, the samples were heated to 95 °C for 5 min and cooled on ice prior loading onto NuPAGE 10% Bis-Tris Gels (life technologies). The whole exosome lysate was loaded onto the gel, this way the four samples were directly comparable since the starting volume was the same. SDS polyacrylamide gelelectrophoresis (SDS-PAGE) was performed according to the manufacturer's specification. Proteins were fixed within the polyacrylamide matrix by incubating the entire gel in 5% acetic acid in 1:1 (vol/vol) water:methanol for 30 min. After Coomassie staining (60 min) the gel slab was rinsed with water (60 min) and each lane was excised and cut into small pieces. Subsequently the proteins were in-gel destained (100 mM ammonium bicarbonate/acetonitrile 1:1 (vol/vol)), reduced (10 mM DTT), alkylated (50 mm Iodoacetamide) and finally Trypsin digested by overnight incubation at 37 °C. The generated peptides were collected from the gel pieces, which were further subjected to a peptide extraction step with an acidic (1.5% formic acid) acetonitrile (66%) solution. Both peptides containing samples are combined and dried down in a vacuum centrifuge.
Nupage 10 bis tris gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
NuPAGE 10% Bis-Tris gels are pre-cast polyacrylamide gels used for protein separation and analysis. They are designed for use with the NuPAGE electrophoresis system. The gels have a Bis-Tris buffer system and a 10% acrylamide concentration.
Automatically generated - may contain errors
Lab products found in correlation
Nupage lds sample buffer, by Thermo Fisher Scientific (7 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Wuhan Servicebio Technology (4 mentions)
Lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Thermo Fisher Scientific (4 mentions)
Hybond, by Cytiva (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Typhoon trio imager, by GE Healthcare (3 mentions)
Iblot, by Thermo Fisher Scientific (3 mentions)
Seeblue plus2 pre stained standard, by Thermo Fisher Scientific (3 mentions)
Odyssey clx scanner, by LI COR (2 mentions)
Immobilon fl pvdf membrane, by Merck Group (2 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (2 mentions)
P0260, by Agilent Technologies (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
95 protocols using nupage 10 bis tris gel
1
Exosomal Protein Extraction and Digestion
2
Clusterin Protein Expression Analysis
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Protein concentrations were determined with a bicinchoninic acid colorimetric assay (BCA) (ThermoScientific). Equal amounts of protein (20 μg) were loaded and separated on NuPAGE® 10 % Bis-Tris gels (Life Technologies). Proteins were transferred onto polyvinylidene difluoride membranes (Hybond P, Amersham Biosciences). After blotting, membranes were blocked in 5 % skimmed milk in PBS containing 0.1 % Tween® 20 (Merck) (PBT) for 2 h at room temperature and probed overnight with a mouse anti-clusterin antibody (1:200, B-5 Santa Cruz recognizing the CLU β–chain) at 4 °C. Equal loading was controlled using a mouse α-tubulin antibody (1:10000, GeneTex). Immunodetection was achieved with horseradish peroxidase (HRP)-conjugated sheep anti-mouse antibody (1:10000, BioSciences) and an ECL Plus™ chemiluminescent detection system (GE Healthcare). Bands were visualized and quantified using ImageQuant LAS 4000 equipment (GE Healthcare Life Sciences).
3
Western Blot Analysis of Protein Lysates
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Proteins were isolated using RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Life Technologies). For Western blot analysis 5 μg of protein lysates were loaded per lane on NuPage 10% Bis-Tris gels (Life Technologies) in 2-(N-morpholino)ethanesulfonic acid (MES) running buffer (Life Technologies). Samples were denatured using 10% mercaptoethanol at 95°C for 10 minutes before loading. Samples were transferred to Immobilon FL PVDF membranes (Millipore) and blocked in Li-cor blocking buffer for 1 hour. Primary antibodies were incubated overnight at 4°C and secondary IRDye antibodies were incubated at room temperature for 1 hour (Licor). The following primary antibodies were used for Western blotting: Actin antibody (Abcam, ab3280), EGF Receptor (Cell Signaling, CS#4267), Phospho-Akt (Ser473) (Cell Signaling, CS#4060), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, CS#4370), N-Cadherin (BD Biosciences, 610921), E-Cadherin (BD Biosciences, 610182) and Cytokeratin 14 (Abcam, ab15461). Near-infrared fluorescence visualization was measured using Odyssey CLx scanner (Li-Cor, Cambridge, UK).
4
Western Blot Protein Quantification
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Protein lysates were acquired using RIPA lysis buffer supplemented with both phosphatase and protease inhibitor cocktails (Life Technologies). For western blots, 5 μg protein was used per lane, unless otherwise stated. Samples were denatured using 10% mercaptoethanol at 95 °C for 5 min and run on NuPage 10% Bis-Tris gels (Life Technologies) in 2-(N-morpholino)ethanesulfonic acid (MES) running buffer. Samples were then transferred to Immobilon FL PVDF membranes (Millipore). Membranes were blocked in Li-cor blocking buffer and primary antibodies were incubated overnight at 4 °C. Near-infrared fluorescence visualization was measured using Odyssey CLx scanner (Li-Cor, Cambridge, UK).
5
Western Blotting Analysis of Cellular Fractions
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For Western blotting analysis, cells were lysed in lysis buffer (50 mM Tris base, pH 7.5, 150 mM NaCl and 1 mM Mg2Cl) supplemented with 1% (vol/vol) Triton X-100, 1 x Protease/phosphatase inhibition cocktail (5872S, Cell Signaling Technologies) and separated by electrophoresis through NuPAGE™ 10% BisTris gels (NP0302BOX or NP0301BOX, Life Technologies). For specific detection of proteins in the cytosol and the nucleus, reagents from the NE-PER Nuclear and Cytoplasmic Extraction Reagent kit (78833, Thermo Fisher Scientific) were used according to the manufacturer’s instructions with few modifications as previously described (19 (link), 20 (link)). Presence and absence of GAPDH was used as marker for the cytosolic and the nuclear fractions, respectively. Proteins were transferred to nitrocellulose membranes (LC2001, Life Technologies) and visualized with primary antibodies and HRP-conjugated rabbit anti-mouse Ig (P0260, DAKO) or HRP-conjugated swine anti-rabbit Ig (P0399, DAKO) with ECL luminescence reagent (RPN2232, Sigma Aldrich) on a ChemiDocTM MP Imaging System (Bio Rad) and subsequently analyzed using the software ImageLab.
6
Western Blot Procedure for Protein Analysis
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Cells were lysed in ice-cold RIPA buffer (Beyotime Biotechnology, Jiangsu, China). The lysates were centrifuged at 4°C for 20 min at 14 000g. The supernatants were retained for the subsequent procedures. The protein concentrations were determined using a BCA protein assay kit (Beyotime, Shanghai, China). The samples were separated on NuPAGE 10% Bis-Tris gels (Life Technologies, Beijing, China) and transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Billerica, Massachusetts, USA). Then, the membranes were incubated overnight at 4°C with a primary antibody and incubated with an HRP-conjugated secondary antibody for 1 h. Then, the membranes were visualized using an ECL Plus Kit (Beyotime Biotechnology).
7
Deglycosylation and SDS-PAGE Analysis of Immunoglobulins
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Twenty microliters of each 0.2 mg/mL Ig sample was mixed with or without 1 μL of PNGase F (New England BioLabs, Ipswich, MA) and incubated overnight at 37°C. Both deglycosylated and glycosylated Ig samples were reduced with 50 mM dithiothreitol (Invitrogen, Carlsbad, CA) at 70°C for 30 min. Reduced and nonreduced samples were then mixed with 4X LDS loading dye (Life Technologies, Grand Island, NY) containing 100 mM iodoacetamide (Life Technologies) and incubated at 100°C for 5 min. Samples were cooled to room temperature and separated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) using NuPAGE 10% Bis-tris gels (Life Technologies) and MOPS running buffer (Life Technologies) at 150 V for 75 min. Gels were stained with Coomassie Blue R-250 (Teknova, Hollister, CA) and destained with 40% methanol and 10% acetic acid. Gels were digitized using an AlphaImager (Protein Simple, Santa Clara, CA) gel imaging system.
8
Western Blot Analysis of PIMT in Mouse Brain
9
Proteomic Analysis of Mammary Organoid Cultures
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Seven biological replicates from each culture condition were subjected to proteomic analysis. Mammary organoid cell pellets were resuspended in 100 μL lysis buffer, which contained 1% sodium dodecyl sulfate (SDS), 75 mM NaCl, 50 mM trishydroxymethylaminomethane (Tris, pH 8.0), and cOmplete mini protease inhibitor mixture (one tablet per 10 mL buffer; Roche). The cells were lysed by sonication (2 s per pulse × 2 pulses, with 15 min on ice between each pulse). Then, the lysates were centrifuged. The volume of the supernatant was reduced using a SpeedVac (0 °C). The protein concentration of each replicate was determined using the bicinchoninic acid (BCA) assay. After volume reduction, an aliquot of each sample (18.75 μL) was loaded onto NuPAGETM 10% Bis-Tris gels (Invitrogen; 1.0 mm, 10 well) and run for 0.5 cm to remove salts in the lysis buffer. The protein bands were cut out of the gels, reduced by incubation with 20 mM Tris 2-carboxyethyl phosphine, alkylated with 40 mM iodoacetamide, and trypsin digested overnight at 37 °C. The resulting peptides were extracted with water/acetonitrile (ACN)/formic acid (FA) (50:50:0.1; vol/vol), dried under vacuum, and resuspended in water/ACN/FA (98:2:0.1; vol/vol) to a final concentration of 0.33 μg/μL.
10
LL-37 Stimulation of HBEC-3KT Cells
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HBEC-3KT cells were stimulated with LL-37, citLL-37, or sLL-37 (2.5 μm each) for 30 min; cell lysates were obtained using ×1 Cell Lysis Buffer (Cell Signaling Technology) containing protease inhibitor cocktail (New England Biolabs). Total protein concentration for each cell lysate sample was determined using a microBCA protein assay kit (Thermo Fisher Scientific, MA, USA). Cell lysates containing 25 μg total protein per sample were used resolved on 4–12% NuPageTM 10% Bis-Tris gels (Invitrogen) followed by transfer to nitrocellulose membranes (Millipore, MA, USA). Membranes were blocked with Tris-buffered saline (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.1% Tween-20) containing 3% bovine serum albumin, followed by probing with phospho-site-specific antibodies obtained from Cell Signaling Technologies for phospho-AKT (T308) and phospho-AKT (473), and anti-human β-actin antibody obtained from Millipore (Burlington, MA, USA) for loading control, in Tris-buffered saline containing 2.5% BSA. The membranes were developed using ECL Prime detection system (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions.
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