For immunoblotting analysis cells were lysed with ice-cold NP-40 Cell Lysis Buffer (Invitrogen) plus protease inhibitors (Roche) for 30 min at 4ºC. Equivalent amounts of protein were first mixed with sample buffer, then loaded on a Criterion Tris-HCl 4–20% pre-cast gel (Bio-Rad) and finally transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA in Tris-buffered saline (pH 7.4) containing 0.05% Tween 20, then incubated with primary and secondary antibodies according to the manufacturer’s instructions. Primary antibodies anti-phospho-p65, and anti-p65 (Cell Signaling Technology) were used, followed by isotype-matched, horseradish-peroxidase-conjugated secondary antibodies (GE Healthcare). Chemi-luminescence detection (Denville Scientific, Inc.) was finally performed.
Criterion tris hcl 4 20 pre cast gel
Manufactured by Bio-Rad
The Criterion Tris-HCl 4–20% pre-cast gel is a laboratory equipment product used for protein electrophoresis. It is a pre-cast polyacrylamide gel with a gradient of 4-20% acrylamide concentration, which allows for the separation and analysis of a wide range of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Np40 cell lysis buffer, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor, by Roche (3 mentions)
Calnexin, by Merck Group (2 mentions)
Anti alix, by Merck Group (1 mentions)
Anti tsg101, by Merck Group (1 mentions)
Anti calnexin, by Merck Group (1 mentions)
Anti cd9, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Tsg101, by Merck Group (1 mentions)
Anti p53, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
4 protocols using criterion tris hcl 4 20 pre cast gel
1
Immunoblotting analysis of phospho-p65
2
Exosomal Protein Analysis by Western Blot
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For the western blot analysis, exosomal proteins were isolated from 500 μl of plasma (the yield of exosomal protein was 9 mg/sample). The exosomal pellet was resuspended in RIPA buffer (Cell Signaling technology), supplemented with phosphatase and protease inhibitors (Roche), and incubated in ice for 20′. Samples were then harvested at 14,000 x g for 10′, and the supernatant collected in a new eppendorf. Protein concentration was determined by using Bradford Assay (Bio-Rad), following the manufacturer’s instructions. 80 μg of exosomal lysate were then loaded on a Criterion Tris-HCl 4–20% pre-cast gel (Bio-Rad), transferred onto a nitrocellulose membrane (Bio-Rad) and probed with anti-Alix (1:1000), anti-TSG101 (1:1000), anti-Calnexin (Sigma) (1:2000), and anti-CD9 (Cell Signaling Technology) (1:1000) primary antibodies, followed by isotype matched, horseradish-peroxidase-conjugated secondary antibodies. Finally, the proteins of interest were detected through chemi-luminescence reaction.
3
Immunoblotting Analysis of Cellular Proteins
4
Immunoblotting Analysis of Cellular Proteins
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For immunoblotting analysis, cells were lysed with ice‐cold NP‐40 Cell Lysis Buffer (Invitrogen) supplemented with protease inhibitors (Roche) for 30 min at 4°C. Equivalent amounts of protein were first mixed with sample buffer, then loaded on a Criterion Tris‐HCl 4%–20% precast gel (Bio‐Rad) and transferred to PVDF or nitrocellulose membranes. Membranes were incubated overnight at 4°C with commercially available antibodies as indicated per experiment: Alix (#SAB4200477, Sigma) calnexin (#C7617, Sigma); CD9 (#D8O1A, Cell Signalling Technology); CD81 (#10630D, Invitrogen), ApoE (#sc‐390925, Santa Cruz), Albumin (#A6684, Sigma). The proteins of interest were detected through chemiluminescence reaction.
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