Streptavidin magnetic beads (200 μg; Dynabeads M-280 Streptavidin (#112-06D); Invitrogen, Carlsbad, CA) were subjected to two rounds of blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 15 min followed by washing with PBS containing 1M NaCl and TE buffer. Biotinylated non-risk or risk oligonucleotides were bound to 100 μL of the BSA-blocked streptavidin beads by incubating for 30 min at room temperature in TE buffer, followed by washing with TE buffer. A biotinylated scrambled oligonucleotide served as a negative control (Supplementary Table 1 ). Fifty micrograms of nuclear extract were pre-cleared by incubating with 100 μL of the BSA-blocked beads in binding buffer (250 mM NaCl, 50 mM Tris Cl, 50% glycerol, 2.5 mM DTT, 2.5 mM EDTA, pH 7.6) containing 15 ng/μL poly dI:dC (#81349-500UG; Sigma-Aldrich, St. Louis, MO), 0.5 μg/mL BSA, and 0.1% NP40 for 30 min on ice. Pre-cleared nuclear extracts were incubated with the oligonucleotide-linked Streptavidin beads for 30 min in a 37 °C water bath. Samples were gently shaken every 5 min. Beads were subsequently washed three times with binding buffer containing 0.1% NP40. The proteins were eluted in 50 μL of 0.2% SDS sample buffer by boiling for 5 min then resolved on a Criterion XT 4%–12% Bis-Tris gel (#3450124; BioRad, Hercules, CA) followed by western blotting.
Criterion xt 4 12 bis tris gel
Manufactured by Bio-Rad
Sourced in United States
The Criterion XT 4%–12% Bis-Tris gel is a pre-cast polyacrylamide gel used for protein separation and analysis. It features a gradient of 4% to 12% acrylamide concentration, which allows for the separation of a wide range of protein molecular weights. The gel is formulated with Bis-Tris buffer, providing a neutral pH environment for protein separation.
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Lab products found in correlation
Hrp coupled secondary antibody, by Jackson ImmunoResearch (2 mentions)
Image lab 5, by Bio-Rad (2 mentions)
Rabbit anti cav 1, by Abcam (2 mentions)
Mouse anti transferrin receptor, by Thermo Fisher Scientific (2 mentions)
Rabbit anti connexin 43 antibody, by Thermo Fisher Scientific (2 mentions)
Mouse anti phospho caveolin1 py14, by BD (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Rabbit anti β catenin, by Abcam (2 mentions)
Ecl plus, by GE Healthcare (2 mentions)
Poly di dc, by Merck Group (1 mentions)
Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions)
Bis tris gel, by Bio-Rad (1 mentions)
Bio safe coomassie g 250, by Bio-Rad (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Bio safe coomassie blue, by Bio-Rad (1 mentions)
Mass spectrometry grade trypsin, by Promega (1 mentions)
Micro bio spin 6 column, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
8 protocols using criterion xt 4 12 bis tris gel
1
Protein-DNA Interaction Profiling
2
Immunoblot Analysis of HA Expression
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To confirm HA expression, 2 g of biomass were homogenized in 4 ml extraction buffer with 1% phenylmethylsulfonyl fluoride. Homogenates were clarified by centrifugation (10,000 × g, 10 min) and the crude extracts (25 µl/sample) were separated a Criterion XT 4–12% Bis-Tris gel (Bio-Rad) under reducing conditions and then transferred onto a PVDF membrane. Successful transfer was confirmed using ponceau red staining followed by de-staining with water. Membranes were blocked overnight (4 °C) with 5% skim milk in TBST (tris-buffered saline, 0.1% Tween 20) and then incubated with rabbit polyclonal anti-H1 (Cat. No. IT-003-SW, Immune Technology) diluted 1:500 in TBST + 2% skim milk for 1 h at room temperature (RT). Membranes were then incubated for 1 h (RT) with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat. No. IT-200-01, Immune Technology) diluted 1:20,000 in TBST + 2% skim milk. Bands were developed using Super Signal West Pico chemiluminescent substrate (Thermo Fisher) and detected on X-ray films. For VLP composition and purity analysis, purified VLP products (5 µg/sample) were separated on a 4–12% Bis-Tris gel as described above followed by staining with biosafe Coomassie G-250 (Bio-Rad). Gels were imaged using ChemiDocTM XRS + system (Bio-Rad). See Supplementary Fig. 11 for unmodified gel and blot images.
3
CD147 Protein Detection via Western Blot
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Cell lysis and immunodetection were performed as described 4 and separation was done using a 4-12% SDS-acrylamide gradient gel (Criterion XT 4-12% bis-tris gel; Bio-Rad, Hercules, CA, USA). Protein extracts of CD3 + cells were further concentrated using Amicon Ultra-2 10 K filter membranes (Millipore, Schwalbach/Ts., Germany) according to the manufacturer's instructions. Immunoreagents used for western blot were the mouse monoclonal CD147 antibody HIM6 (1:100; BD Bioscience) and the mouse monoclonal anti-α-Tubulin (1:5000; Sigma) as loading control.
4
Plasma Membrane Protein Analysis
5
Super-SILAC Proteomics Protocol
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Unlabeled primary cell lysates were mixed with the super-SILAC standard in a 1:1 ratio by protein content, as determined by BCA assay. All samples were desalted and buffer exchanged to Tris/HCl buffer using Micro bio-spin 6 columns (Bio-Rad, Hercules, CA). Protein concentration was determined by BCA assay and aliquots containing 80ug of total protein per sample run (up to 240ug for triplicate analysis) were dried by vacuum centrifugation. Protein pellets were resuspended in XT sample buffer containing Criterion XT reducing agent and denatured at 95°C for five minutes. Proteins were resolved by SDS-PAGE on Criterion XT 4-12% Bis-Tris gels (Bio-Rad, Hercules, CA) at 180 volts for 1 hour. The gels were fixed (50:5:45/methanol:aceticacid:water/v:v:v), stained with Bio-Safe Coomassie blue (Bio-Rad, Hercules, CA) and destained in H2O. 32 gel sections were excised from each sample run and the individual bands were processed for in-gel trypsin proteolysis as previously described10. Trypsin digestion was performed using 12.5ng/ul of mass spectrometry grade Trypsin (Promega, Madison, WI) diluted in 25mM NH4HCO3 solution. Peptides recovered from each band were dried by vacuum centrifugation and resuspended in 8ul of 0.1% TFA for mass spectrometry analysis.
6
Plasma Membrane Protein Analysis
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Three confluent 160-mm-diameter dishes of MBECs were harvested and plasma membranes were isolated as previously described (Yao et al., 2009). Protein concentrations were determined by Bio-Rad Dc-Protein assay (Bio-Rad). Samples were mixed with Bio-Rad XT sample buffer and reducing agent, run on Criterion™ XT 4–12% Bis-Tris Gels (Bio-Rad), and transferred onto PVDF membranes. Immunodetection was performed with (1:100) rabbit anti-connexin 43 antibody (Invitrogen), (1:500) mouse anti phospho-caveolin1 (pY14) (BD Transduction Laboratories), (1:5000) rabbit anti-β-catenin (Abcam), (1:1000) rabbit anti-Cav-1 (Abcam), or (1:1000) mouse-anti-transferrin receptor (Life Technology) followed by HRP-coupled secondary antibodies (Jackson ImmunoResearch), and developed with ECL-Plus (GE Healthcare). Protein bands were quantified by densitometry using Image Lab 5.1 (Bio-Rad) software. Plasma membrane connexin43 levels were normalized to plasma membrane transferrin receptor. This value was normalized to total β-catenin as a housekeeping reference protein.
7
Verification of IPNV Protein Expression
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Expression of IPNV proteins in cell cultures was also verified by Western blots of lysate of transfected cells or cell culture medium. The cells or cell culture medium were dissolved in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and the proteins were separated on a Criterion XT Bis-Tris gel 4%–12% (Bio-Rad; Hercules, CA, USA), with XT-MOPS as running buffer and blotted onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) following the Criterion™ Precast Gel system protocol (Bio-Rad). The blot was incubated with polyclonal anti-IPNV (1:5000) or anti-VP3 antibody (1:1000) overnight followed by HRP conjugated anti-rabbit IgG antibody and anti-mouse IgG, respectively, for 2.5 h, and 5% non-fat milk in PBS-SIFF (PBS in 0.1% Tween-20) were used as blocking solution. The membranes were then incubated with substrate from ECL Plus™ Western Blotting (GE HealthCare, Cleveland, OH, USA) for 5 min and detected with ChemiDoc XRS (Bio-Rad).
8
Protein Expression and Immunoblotting Analysis
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HPMEC or monocytes were lysed in PhosphoSafe Extraction Reagent (Novagen, Merck Biosciences, Nottingham, U.K.) and centrifuged for 5 min at 16,000 g at 4°C to collect the supernatant. Protein concentration was determined (BCA Protein Assay Kit, Pierce; ThermoScientific), and thirty micrograms of each sample were electrophoresed on a Criterion XT Bis-Tris Gel 4–12% (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Millipore, Saint-Quentin en Yvelines, France). The membrane was blocked with 5% w/v skim milk powder in TBST (0.1 M Tris-HCl pH 8,1.5 M NaCl and 1% Tween-20) for 2 h at room temperature, and subsequently incubated with anti-TREM-1 (AbD Serotec), anti-(p)ERK1/2, anti-(p)eNOS, anti-(p)P65 (Nuclear Factor-κB p65), and anti-His (Cell Signaling, USA) antibodies overnight at 4°C. After vigorous washing in TBST, the membrane was incubated with a secondary antibody conjugated to horseradish peroxidase for 1h at room temperature. Immunocomplexes were detected with the SuperSignal West Femto Substrate (Pierce; ThermoScientific). Non-phosphorylated forms or tubulin (Cell Signaling) were used for normalization. Acquisition and quantitative signal density analyses were performed by a LAS-4000 imager (FSVT) and Multi-Gauge software (LifeScience Fujifilm, Tokyo, Japan).
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