Fresh retinal extracts were solubilized in Nonidet P-40 buffer using Bullet Blender BBX24 (Next Advance Inc.) in the presence of 0.5mm zirconium oxide beads (Next Advance Inc., Averill Park, NY). 1.2 mg of retinal extracts were pre-cleared with 2μg of non-immunized IgG (Stressgen, San Diego, CA) and incubated with 50μl of 50% protein A/G bead slurry (Santa Cruz Biotechnology) at 4°C for 1 hour. The supernatants were incubated with 2μg of mouse anti-HA antibody (Abcam, Cambridge, MA) at 4°C for 12 hour. Co-immunoprecipitate complexes were resolved by SDS-PAGE in 4–15% gradient Criterion gels (BioRad) and immunoblotted with mouse anti-Pttg1 antibody (Abcam) as described [33 (link),48 (link)].
Criterion gradient gels
Manufactured by Bio-Rad
4–20% Criterion gradient gels are laboratory equipment designed for protein separation and analysis. They provide a continuous gradient of polyacrylamide concentrations, ranging from 4% to 20%, allowing for the effective separation of a wide range of protein molecular weights in a single gel.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot sd cell, by Bio-Rad (2 mentions)
Reliablot kit, by Fortis Life Sciences (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Complete protease inhibitor cocktail, by Merck Group (2 mentions)
Bullet blender bbx24, by Next Advance (1 mentions)
0.5 mm zirconium oxide beads, by Next Advance (1 mentions)
Mouse anti ha antibody, by Abcam (1 mentions)
Chemiluminescence reagent, by Cytiva (1 mentions)
Goat anti rabbit, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Peroxidase coupled goat anti rabbit igg, by Merck Group (1 mentions)
Nu page lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Image studio 2, by LI COR (1 mentions)
Histone h3, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Immobilon fl pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
8 protocols using criterion gradient gels
1
Co-immunoprecipitation of Pttg1 Protein
2
Western Blot Analysis of Microglia
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The collected microglial samples were homogenized with Laemmli Sample buffer (BioRad), loaded onto 4–15% gradient Criterion gels (Bio-Rad) followed by electrophoretic transfer to Nitrocellulose membranes (Bio-Rad) using a standard protocol. Samples were normalized to 1 µg/ml and the loading volume was 20 µl/well. The membranes were incubated with rabbit polyclonal CD11b (2 µg/ml, Abcam) and mouse anti-β-actin (1∶5000, Sigma) over night at 4°C, followed by reactions with respective goat anti-rabbit (1∶5000) and –mouse (1∶5000) (Sigma) peroxidase conjugates. The protein bands for CD11b and β-actin were visualized by chemiluminescence reagents (Amersham, Piscataway, NJ) and quantified by Image J software.
3
Protein Extraction and Western Blotting
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Cells were lysed in 1x RIPA buffer (1% NP-40, 0.1% SDS, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium deoxycholate, 1 mM EDTA) for whole cell extracts, or 1x EBC buffer (120 mM NaCl, 0.5% v/v NP-40, 50 mM Tris-HCl pH 8.0) for immunoprecipitations, 0.5 μM DTT (0.01% 2-Mercaptoethanol for EBC buffer), 25 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and cOmplete protease inhibitor cocktail (Sigma). PVDF membranes were blocked in 5% milk/0.1% Tween 20 TBS for 1 hr at room temperature. Primary antibodies were incubated overnight at 4°C. After incubation with either the secondary IRDye Alexa Fluor 680 goat antimouse antibody or 800 goat anti-rabbit antibodies (Odyssey), the membranes were visualized with the Odyssey Infrared Imaging System (Li-Cor). Immunoprecipitations: whole cell lysates (10% inputs) and immunoprecipitates were resolved on 4%–15% gradient Criterion gels (BioRad) and transferred to nitrocellulose membranes using semi-dry Transblot SD cell (BioRad). ReliaBLOT kit (Bethyl Labs; WB120) was used for IP/western blotting according to manufacturer’s protocol and membranes were probed with indicated antibodies.
4
Protein Extraction and Western Blotting
5
GC Protein Extraction and SDS-PAGE
6
Protein Extraction and Western Blotting
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Total seedling or inflorescence protein samples were extracted from 100 mg of ground tissue with NuPAGE lithium dodecyl sulfate sample buffer (Invitrogen) according to Ref. 69 (link). Equal volumes of extract from different samples were separated on precast 4–20% Criterion gradient gels (Bio-Rad) before transfer to nitrocellulose membranes (Amersham Biosciences Protran Premium 0.45 μm, GE Healthcare) using a trans-blot blotting apparatus (Bio-Rad). Primary antibodies were detected using peroxidase-coupled goat anti-rabbit IgG (Sigma) and visualized using chemiluminescence SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
7
Quantification of Calcineurin Levels
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CN levels were assessed in nuclear and cytosolic fractions as described in our earlier work (Mohmmad Abdul et al., 2011 (link); Sompol et al., 2017 (link)). Half brains from CT and HHcy diet mice were harvested and stored at −80°C as described above. Cytosolic and nuclear homogenate samples were resolved via SDS PAGE on 4–20% Criterion gradient gels (Bio-Rad) and then transferred to Immobilon-FL PVDF membranes (Millipore Sigma). Membranes were probed for calcineurin anti-CN-Aα (catalog #07-1492, EMD Millipore), which tags the N-terminal region of CN and identifies both full-length phosphatase and C-terminal proteolized phosphatase fragments (Mohmmad Abdul et al., 2011 (link)). GAPDH (catalog #ab9484, Abcam) and H3 Histone (catalog #H0164, Sigma-Aldrich) were used to mark cystolic versus nuclear fractions, and βActin (catalog #3700S, Cell Signaling Technology) was used to normalize CN signal as an internal control. Membranes were then imaged on an Odyessey Scanner (LI-COR) and the quantification conducted using Image Studio 2.1 software (LI-COR).
8
Western Blot Analysis of HepG2 Cells
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Proteins from HepG2 cells were extracted at 4°C using an ice-cold lysis buffer (10 mM Tris-HCl, 150 mM NaCl2, 2 mM EDTA, 1% Triton X-100, 10% glycerol and 1 mM DTT), supplemented with protease and phosphatase inhibitors (Sigma Aldrich). 20–30 micrograms of protein extracts were separated by SDS-Page using 4–20% Criterion gradient gels (Bio-Rad Laboratories, Richmond, CA) and transferred to nitrocellulose membranes. After blocking, the membranes were probed overnight at 4°C with primary antibodies purchased from Cell Signaling Technology (Danvers, MA, USA) (p-Akt), Sigma-Aldrich (anti-GAPDH) and Abcam (MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail, Abcam, Cambridge, MA, USA). Odyssey anti-rabbit IRDye 800CW and anti-mouse IRDye 680RD (LI-COR Biosciences, Lincoln, NE, USA) were used as secondary antibodies. Finally, the blots were scanned and quantified by using Odyssey CLX Infrared Imager of Li-COR and manufacturer's software. If re-probing was needed, the membranes were incubated for 10 min in 0.2 M NaOH at RT, washed with TBS and re-probed with appropriate antibodies. All samples and results were normalized to GAPDH.
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