Bovine CMEC whole cell lysates from cells grown for 48 h in ACM containing medium and freshly isolated rat brain microvessel lysates were subjected to Western blot analysis. Rat brain microvessels were isolated as described.25 (link) Five mg protein from each sample was denatured with 50 mM dithiothreitol (DTT) in SDS sample reducing buffer (NP0004, Life Technologies) and heated at 70°C for 10 min. Protein samples were then separated on 12% Tris-HCl gel (No.5952, PAGEr™ Gold Precast Gel, Lonza) using electrophoresis and electrotransferred to polyvinylidene fluoride membranes (BioRad XCell SureLock, Hercules). Membranes were blocked with 7.5% milk in PBS with 0.1% Tween-20 (PBST) for 1hr, incubated with anti-KCa3.1 antibody (AV35098, rabbit polyclonal, 1:3000, Sigma-Aldrich, Louis, MO) in milk/PBST overnight at 4°C, washed with PBST, and incubated with horseradish peroxidase-conjugated secondary antibody (A16096, goat anti-rabbit, 1:2000, Life Technologies) in milk/PBST for 1 hr at room temperature. After washing with PBST, bound antibody was detected using enhanced chemiluminescence kit (RPN2133, ECL plus; GE Healthcare) and visualized on a Fuji Film LAS-4000 Imaging Machine (Medford, UK).
Pager gold precast gel
Manufactured by Lonza
Sourced in United States
PAGEr Gold Precast Gels are a type of laboratory equipment used for electrophoresis, a technique that separates molecules based on their size and charge. These precast gels are designed to provide a reliable and consistent platform for protein or nucleic acid separation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Np0004, by Thermo Fisher Scientific (1 mentions)
Las 4000, by Cytiva (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Halt protease phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Lide 210 scanner, by Canon (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Odyssey infrared scanner, by LI COR (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions)
Iblot gel transfer stacks nitrocellulose, by Thermo Fisher Scientific (1 mentions)
Anti pten, by Abcam (1 mentions)
Anti erg, by Abcam (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay reagent, by Bio-Rad (1 mentions)
Anti mouse or anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
6 protocols using pager gold precast gel
1
Western Blot Analysis of KCa3.1 in Brain Microvessels
2
Kidney Protein Extraction and Western Blot
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Approximately 100 mg of frozen kidney was taken and pulverized to a fine powder, suspended in 900 µl of T-PER (Pierce, Rockford, IL, USA) containing 1 % protease and phosphatase inhibitors and homogenized. Supernants were collected by centrifugation (10,000×g for 10 min at 4 °C and the protein concentration determined using the 660 nm assay (Pierce, Rockford, IL, USA). Samples were equally diluted with 4× Laemilli buffer and then subjected to electrophoresis on 10 % PAGEr Gold Precast gel (Lonza, Rockland, ME, USA) before transfer to nitrocellulose membranes as detailed elsewhere [44 (link)]. Equal loading of protein was verified by Ponceau S staining of nitrocellulose membranes. Membranes were blocked with 5 % milk in TBST for 1 h and later probed with primary antibodies against p-stat-3 (Tyr 705), stat-3, cleaved caspase-3 and caspase-3 (Cell Signaling Technology, Danvers, MA, USA). After exposure to the primary antibodies, membranes were washed with TBST (3 × 5 min) and incubated with secondary anti-rabbit (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. Immunoreactivity was visualized using Supersignal West Pico Chemiluminiscent substrate (Pierce, Rockford, IL, USA) and quantified by Fluorchem 9900 software (Protein Simple, Santa Clara, CA, USA).
3
CFSE-labeled OT-I CTLs Isolation and Western Blot
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CFSE-labeled OT-I CTLs were isolated using fluorescence-activated cell sorting (FACS) from unlabeled EL-4 and aTreg in co-culture experiments. For protein extraction, cells were lysed in RIPA buffer (Thermo Scientific, #89900) and 1 × Halt protease & phosphatase inhibitor cocktail (Thermo Fisher Scientific, #78440). Electrophoresis was performed on PAGEr Gold Precast Gels (Lonza, #58522), and proteins were transferred to nitrocellulose membranes (Thermo scientific, #88018). After blocking with 5% nonfat dry milk, the blots were incubated overnight with the different primary antibodies used. All secondary antibodies were conjugated with horseradish peroxidase (HRP). SuperSignal West pico (Thermo scientific, #32106) and Supersignal West Femto (Pierce, #34095) were used to detect HRP on immunoblots with X-ray film (Pierce, #34090). Films were scanned using a LiDE 210 Scanner (Canon). Important uncropped western blots are shown in Supplementary Fig. 7 .
4
Western Blot for Cardiomyocytes and Zebrafish
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For rat cardiomyocytes, protein was extracted using cell lysis buffer (Cell Signaling) with 1 mM PMSF (Sigma) and then subjected to sonication. The protein concentration was determined by Dc protein assay (Bio-Rad). For zebrafish, 15 embryos were suspended directly in 50-μl SDS loading buffer and homogenized using a tissue homogenizer (TissueLyser II, Qiagen). Following homogenization, the samples were centrifuged and total protein homogenates were obtained. 30 μg of total protein or 18 μl of fish protein lysate was loaded onto Criterion XT bis-tris precast gels (4–12%) (Invitrogen) or PAGEr Gold precast gels (4–20%) (Lonza) for electrophoresis. Protein was electrotransferred to a PVDF membrane (Millipore) at 30 volts for 16–18 h at 4°C. After blocking in 5% BSA in PBS, proteins of interest were detected by incubation with appropriate primary antibodies overnight at 4°C. After washing, blots were incubated with corresponding secondary antibodies. Odyssey infrared scanner (Li-Cor) was used to determine the infrared fluorescent signal, and GAPDH was used as a reference gene for normalization.
5
Western Blot Analysis of ERG, PTEN, and β-Actin
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Cells were lysed in modified RIPA buffer (Santa Cruz) supplemented with PMSF, protease inhibitor and sodium orthovanadate. Protein concentration of the lysates were determined using BCA protein assay reagent (Bio-Rad); 30μg of the extracted protein was mixed with Laemmli sample buffer containing β-ME, denatured, separated using 10% PAGEr Gold Precast Gels (Lonza), and transferred using iBlot Gel Transfer system (Invitrogen) and iBlot Gel Transfer Stacks Nitrocellulose (Invitrogen). Anti-ERG (#2805-1, Epitomics), anti-PTEN (#S-0271, Epitomics), anti-β-actin antibody (Santa Cruz Biotech), antibodies were used at 1:1000 dilution with blocking with 5% skim milk. After incubation with primary antibodies overnight at 4°C, horseradish peroxidase–labeled secondary antibodies were then applied to the membranes for 1 h at room temperature. Signals were visualized using ECL Western blotting detection substrate (Thermo Scientific).
6
Immunoblotting of Protein Samples
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Immunoblotting was performed according to standard techniques. Briefly, protein was diluted to 4 mg/mL in Laemmli buffer. Forty micrograms of protein was electrophoresed on 4–20% PAGEr Gold Precast Gels (Lonza, Walkersville, MD) at 120 V by SDS‐PAGE and transferred onto nitrocellulose membranes (Bio‐Rad, Hercules, CA) at 100 V for 1 h at 4°C. To ensure equal loading, membranes were stained with Ponceau‐S stain and imaged with FOTO/Analyst PC Image software. The resulting signal was quantified and was similar between groups for all membranes. Membranes were destained in 1% TBST (50 mmol/L Tris‐HCl, pH 7.4, 150 mmol/L NaCl, 0.1% Tween 20) for 20 min, blocked in 5% milk in 1% TBST for 1 h, and then exposed to primary antibodies overnight at 4°C (Table 1 ). Membranes were then washed, exposed to appropriate anti‐mouse or anti‐rabbit secondary antibodies (Cell Signaling Technology, Danvers, MA) for 1 h at room temperature, washed in 1% TBST, and incubated with ECL Western Blotting Substrate for 5 min at room temperature (Thermo Fisher Scientific, Inc.). Blots were imaged on X‐ray film (Phenix Research Products, Candler, NC), and the resultant bands quantified with Carestream software. Signal from each band was normalized to mean signal from the control (thermal neutral) group.
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