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23 protocols using nupage novex system

1

Western Blotting of Adenosine Receptors

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Cells washed in ice cold PBS buffer then lysed directly in 2XLDS sample buffer (Life Technologies) supplemented with 10 mM TCEP, then heated at 70°C for 10 minutes. Western blotting was performed using the Novex NuPAGE system (Life Technologies) under reducing conditions with 4-12% Bis-Tris gradient gels using MES buffer following the recommended protocol. Protein transfer to nitrocellulose for blotting was performed using the iBlot system (Life Technologies). Blocking and all antibody incubations were performed in 0.25%/0.25% w/w BSA/non-fat dried milk (Marvel) in Tris buffered saline/0.05% Tween-20 (TBST). After 1 hour in blocking buffer, after three brief washes in TBST, primary antibody incubations were performed overnight at 4°C using 1:5000 anti-actin (SCBT, sc-47778), anti-A1R (Proteintech, 55026-1-AP), and antibodies against human adenosine A2A, A2B and A3 receptors (Abcam ab3461, ab40002 and ab136051, respectively,). Membranes were washed in TBST three times for 10 minutes and then incubated with the appropriate 1:10,000 HRP-conjugated secondary antibody (Sigma-Aldrich) for 60 min. Four 10-min TBST washes were then performed before performing chemiluminescence detection using Immobilon ECL reagent (Merck Millipore).
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2

Analysis of Selenoprotein P by Mass Spectrometry

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Purified SelP samples were reduced with DTT, followed by alkylation of Cys and Sec residues with iodoacetamide as described [16 (link)]. Alkylated proteins were resolved by SDS-PAGE using Novex NU-PAGE system (Life Technology) and stained with Coomassie blue. Protein bands corresponding to SelP (individual samples representing the conditions analyzed in the current study) were cut out and subjected to in-gel tryptic digestion and LC-MS/MS analysis. In-gel trypsin digestion of the destained protein bands was carried out for 14 h at 37°C. The resulting peptide mixture was extracted from the gel slices and loaded into a fused silica microcapillary packed with Magic C18AQ beads (Michrom Bioresources). Reversed phase liquid chromatography was performed using an Agilent 1100 pump and a Famous autosampler (LC Packings). Peptides were eluted from the column with a 90-min acetonitrile gradient and detected using an LTQ-Orbitrap Velos (Thermo-Fisher Scientific).
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3

Protein Extraction and Immunoblotting Procedure

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Cells were washed with ice-cold Phosphate-Buffered Saline (PBS, Life Technologies) and collected in RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% IGEPAL, 1% sodium deoxycholate, 0.1% SDS) (Pierce); supplemented with 1X Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific). Samples were incubated on ice for 10 min and cleared by centrifugation at 14000 g for 10 min at 4°C. The protein concentrations of samples were determined with the DC Protein Assay (BioRad) according to the manufacturer's recommendations. Samples were stored at −20°C.
SDS-PAGE and immunoblotting was performed with the Novex NuPAGE system (Life Technologies) according to the manufacturer's recommendations. The protocol is described in detail elsewhere [20] (link). The following primary antibodies were used: anti TP53 central parts (Pab240, diluted 1∶200, Santa Cruz Biotechnology), anti TP53 N-terminal (DO-1, diluted 1∶200, Calbiochem Merck), anti TP53 C-terminal (Pab421, diluted 1∶200, Calbiochem Merck), anti DDIT3 (15204-1-AP, diluted 1∶266, Proteintech) and anti GAPDH (mAbcam 9484, diluted 1∶200, Abcam)
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4

Fluorometric Assessment of Cathepsin Enzymes

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Cathepsin B, L and D activities were assessed fluorometrically using the following substrates: Z-Arg-Arg-AMC (Bachem, Bulbendorf, Switzerland), Z-Phe-Arg-AMC (Bachem) and 7-methoxycoumarin-4-acetyl-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-DNP-D-Arg-amide, as described previously [32 (link)]. LAL was determined by using the pro-fluorescent substrate 4-methylumbelliferyl oleate [33 (link)]. Western blotting was used to assess changes in the protein expression of cathepsin B, L and LAMP-1, following lysis of the cells in water at 4°C, and electrophoresis (90 min, 130 V) using 4–12% bis-tris gels (Novex Nupage system, Life Technologies, Carlsbad, CA, USA) with protein transfer (20 V, 7 min) to a PDVF membrane (iBlot 2, Life Technologies). And incubation with either anti-cathepsin B goat polyclonal (Santa Cruz Biotechnology, Dallas, Texas, USA), anti-cathepsin L mouse monoclonal (Abcam, Cambridge, UK), anti-LAMP-1 rabbit polyclonal (Abcam) or anti-β-actin mouse monoclonal (Santa Cruz) primary antibodies (1/1000 dilution). Proteins were visualized using a ChemiDoc XRS (Bio-Rad, Hercules, CA, USA), following exposure of membranes to chemiluminescence reagents (Western Lightening Plus-ECL, Perkin Elmer, Waltham, MA, USA), with densitometry performed using ImageJ software (National Institutes of Health, USA).
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5

Western Blot Protein Analysis Protocol

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Total protein was extracted using the RIPA buffer (Pierce, Rockford, IL) in the presence of a Protease Inhibitor Cocktail (Pierce). After quantified by the bicinchoninic acid method (Waltham, MA), 80 μg protein was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) using the Novex NuPAGE system (Invitrogen) and denatured for 5 min. Subsequently, the protein was transferred to 0.45‐Am nitrocellulose membranes. Membranes were blocked in TBS‐T buffer containing 5% nonfat milk for 1 h and incubated overnight with the following primary antibodies rabbit anti‐human DLGAP1 and GAPDH (1:800; Abcam, Cambridge, MA). After washed using TBST, the membranes were then hybridized with the horseradish peroxidase (HRP)‐linked antibody goat anti‐rabbit IgG (1:2000; Abcam) for 1 h. Signal detection was carried out with an ECL system (Amersham Pharmacia, Piscataway, NJ).
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6

Mitochondrial Proteome Analysis by Western Blot

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Cells were lysed and separated via the Novex NuPAGE system (Invitrogen, NP0008, NP0001, and LC3675) and the ExpressPlus PAGE Gel (GenScript). Polyvinylidene difluoride membranes (Millipore, IPVH304F0) were used for transfer and then probed with the following primary antibodies in 3% bovine serum albumin/Tris-buffered saline with 0.1% Tween 20 detergent (TBST): β-tubulin (Santa Cruz Biotechnology, sc-23949), adenosine triphosphate synthase 5 alpha (ATP5a) (Abcam, ab14748), Cyt C (Santa Cruz Biotechnology, sc-13156), MFN1 (Abcam, ab57602), MFN2 (Abcam, ab56889), and β-actin (Santa Cruz Biotechnology, sc-47778). The antibodies were detected using horseradish peroxidase–conjugated antibodies (Invitrogen, 31430), the ECL Prime Western Blotting System (GE HealthCare, RPN2232), and the Syngene Pxi Imager.
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7

Protein Expression Analysis in Cartilage Cells

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The cells in the culture were lysed using the RIPA buffer (Pierce, Rockford, IL, USA) with a protease inhibitor cocktail (Pierce). Total protein was separated by SDS-PAGE electrophoresis using the Novex Nu PAGE system (Invitrogen) and transferred to 0.45-μm PVDF membranes. Membranes were blocked in TBS-T buffer containing 5% nonfat milk for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-Collagen X (1:1,000, Abcam, #ab182563), anti-DLX5 (1:1,000, Abcam, #ab109737), anti-RUNX2 (1:1,000, Abcam, #ab264077), anti-SOX9 (1:1,000, Abcam, #ab185966), anti-MMP13 (1:1,000, Abcam, #ab51072), and anti-β-ACTIN (1:1,000, Beyotime, China, #AF0003) was used as the internal control. Then the membranes were washed using TBST and hybridized with the horseradish peroxidase (HRP)-linked antibody goat anti-rabbit IgG (1:4,000, Beyotime) or goat anti-mouse IgG for 1 h. Signal detection was carried out with an ECL system (Amersham Pharmacia, Piscataway, NJ, USA).
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8

Protein Purification and Quantification

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Samples for SDS-PAGE and protein quantification were prepared by using Zeba Micro Spin desalting columns 7 MWCO (Thermo Scientific) following the manufacturer’s protocol and eluted in distilled water. Further processing for SDS-PAGE was performed according to the NuPage Novex system (Invitrogen). NuPage Novex 1.0 mm BisTris 10–12% acrylamide precast gels (Invitrogen) were used for the PAGE analysis. The SeeBlue Plus2 Prestained Standard (Invitrogen) was used as a molecular weight marker. Protein concentrations were determined by the BCA assay using the Micro BCA Protein Assay Kit (Thermo Scientific), using Bovine Serum Albumin as standard.
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9

Western Blot Analysis of Pericentrin

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The levels of expression of pericentrin were determined by Western blot analysis using a rabbit pericentrin polyclonal antibody (ab4448, Abcam Ltd., Cambridge, UK) and the beta actin (A2066, Sigma-Aldrich, Inc., St. Louis, MO, USA) for normalization. The cells washed with PBS buffer plus 0.1 mM Na3VO4 were pelleted and lysed in Laemmli buffer (0.125 M Tris–HCl pH 6.8, 5% SDS) containing protease inhibitors. Lysates were boiled for 2 min, sonicated, and quantitated by Bradford assay. Aliquots containing 30 mg/mL of protein plus 5% b-mercaptoethanol were size fractionated on 3–8% Tris–acetate gel, using the NuPage Novex system from Invitrogen (Carlsbad, CA, USA). After incubation with a peroxidase-conjugated secondary antibody, the immunoreactive bands were visualized by ECL Supersignal on autoradiographic films.
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10

Characterization of Anti-BLG Antibodies

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Binding of purified chimeric human IgE and IgG1 anti-BLG antibodies clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 was done by using the BLG ELISA as described above. For detection, however, 1∶1,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) and 1∶5,000 diluted HRP-conjugated goat anti-human IgG-specific antibodies (Jackson ImmunoResearch) were applied.
Bovine BLG (120 µg/480 µl non-reducing sample buffer) was loaded and electrophoresed in a one well pre-casted 4–12% BisTris gel/MOPS running buffer NuPage Novex system (Invitrogen). Then, bovine BLG was electro-blotted onto a polyvinylidene fluoride transfer membrane (Millipore). After blocking with PBS/0.05% Tween 20/1% BSA fraction V (Roche) for 1 hour at RT, this membrane was incubated with 1 µg/ml purified chimeric human IgE anti-BLG antibody clones 5D6.1, 7D5.1, 8C3.3, 8F7.1, 11B6.2 or 13A5.2 for 1 hour at RT. After washing in PBS/0.05% Tween 20, binding of these chimeric human IgE anti-BLG antibodies was determined with 1∶4,000 diluted HRP-conjugated goat anti-human IgE-specific antibodies (Sigma) for 1 hour at RT, followed by a ready-to-use solution of TMB substrate (Sigma) for colorimetric detection.
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