Cells were harvested and washed twice in ice cold PBS (0.5% FBS) and resuspended in RIPA lysis and extraction buffer (Thermos Fisher) supplemented with Protease and phosphatase inhibitor cocktail (Thermo fisher) at a concentration of 1xe6 cells per 150 ul. The suspension was incubated on ice for 15 minutes with gentle rocking and centrifuged at 14000 RPM for 15 minutes. 120 ul of clear lysates were transferred to new tubes into which 60 ul of Lammli sample buffer (biorad) was added. Tubes were vortexed briefly and boiled at 95 degrees for 10 minutes. For WB analysis, samples were loaded and resolved on 4 - 15% SDS-PAGE precast gels (biorad), transferred to PVDF membranes, blocked in 5% milk in TBS with 0.1% Triton X-100 (Sigma) for 1 hr. After blocking, the PVDF membranes were rinsed briefly with TBST and incubated with primary antibodies diluted in 5% BSA/TBST at 4°C overnight, washed (5 times 10 minutes each), incubated with secondary antibodies at room temperature for 1 hour at 1:10,000 dilution and washed again and finally subjected to imaging. The WB results were imaged on the ChemiDoc Imaging System (Biorad) and visualized in the Image Lab software (BIO-RAD).
4 15 sds page precast gels
Manufactured by Bio-Rad
Sourced in United States, Italy
4 - 15% SDS-PAGE precast gels are polyacrylamide gel electrophoresis (PAGE) products designed for the separation and analysis of proteins. These precast gels are formulated with a gradient of 4% to 15% polyacrylamide, which allows for the effective separation of a wide range of protein molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Lammli sample buffer, by Bio-Rad (2 mentions)
Pierce coomassie bradford protein assay kit, by Thermo Fisher Scientific (1 mentions)
Las 500 image system, by GE Healthcare (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
4 protocols using 4 15 sds page precast gels
1
Western Blot Protein Extraction and Analysis
2
Quantitative Western Blot Analysis
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Protein concentration in the lysates was determined using the Bradford assay with bovine serum albumin as a reference standard (Pierce™ Coomassie Bradford Protein Assay Kit, Thermo Fisher Scientific). Samples containing equal protein amounts in sample buffer were loaded on 4–15% SDS-PAGE precast gels (Bio-Rad, Hercules, CA, USA) and separated by electrophoresis. After proteins were transferred onto polyvinylidene fluoride membrane, the membranes were blocked with blocking buffer (Tris-buffered saline with 5% non-fat dry milk and 0.01% Tween-20) for 1 h at RT. Then, the membranes were incubated with each primary antibody solution (1:1000 in blocking buffer) at 4 °C overnight. The membranes were washed with Tris-buffered saline with 0.01% Tween-20 and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 in blocking buffer). Protein signals were visualized using an enhanced chemiluminescence detection reagent. Protein band images were obtained using an LAS 500 image system (GE Healthcare, Chicago, IL, USA). Blot signals were quantitated by densitometry analysis using ImageJ software (NIH, Bethesda, MD, USA). Protein levels were normalized to those of the corresponding loading controls.
3
Western Blot Protein Extraction and Analysis
4
Synaptosomal Protein Immunoprecipitation
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50 μg of proteins from the crude synaptosomal (P2) fractions were incubated for 1 hour at 4 °C in RIA buffer (200 mM NaCl, 10 mM EDTA, 10 mM Na2HPO4, 0.5% Nonidet P-40 supplemented with 0.1% SDS) and protein A/G-agarose beads (Santa Cruz, Dallas, TX, USA) as pre-cleaning procedure. The beads were then let to sediment at the bottom of the tube and the supernatant was collected. Primary antibody (Homer1bc) was added to the supernatant before leaving to incubate overnight (O/N) at 4 °C on a rotating wheel, then protein A/G-agarose beads were added and incubation continued for 2h at RT. Beads were then collected by gravity and washed three times with RIA buffer before adding sample buffer for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and heating the mix al 95°C for 10 min. Beads were pelleted by centrifugation an supernatants were separated using 4-15% SDS-PAGE precast gels (Biorad, Italy) (Mellone et al., 2015) (link).
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