Cellular proteins were harvested in RIPA lysis buffer containing vanadate, phosphatase inhibitor and phenylmethanesulfonyl fluoride. Proteins were resolved by electrophoresis in polyacrylamide gels and transferred to a nitrocellulose membrane using a Trans-Blot system (Bio-Rad) as described previously [14 (link)]. Membranes were blocked in 5% skim milk diluted with Tris-buffered saline and Tween 20 (TBST) for 1 h at room temperature and incubated with primary antibodies diluted in 3% skim milk in TBST for 1 h at room temperature or overnight at 4°C. Antibodies against the following proteins were used for Western blot analysis (all from Santa Cruz Biotechnology): GRP78 (C–20) (diluted 1:1000), PERK (1:1000), p-PERK (1:250–500), p-eIF2α (1:250–500), GADD153 (1:500), CREB-2 (1:500), and vinculin (1:5000). Anti-goat, anti-rabbit, and anti-mouse IgG secondary antibodies (1:5000) were used. Amersham enhanced chemiluminescence reagents (GE Healthcare) or Supersignal chemiluminescence reagents (Thermo Scientific) were used to detect immunoreactive proteins.
Amersham enhanced chemiluminescence reagent
Manufactured by GE Healthcare
Sourced in United Kingdom
Amersham enhanced chemiluminescence reagents are a set of solutions used in the detection and quantification of proteins in Western blot analysis. The reagents generate a luminescent signal proportional to the amount of target protein present, allowing for sensitive and accurate protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot system, by Bio-Rad (1 mentions)
Super signal chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Grp78 c20, by Santa Cruz Biotechnology (1 mentions)
P perk, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Multi gauge v3, by Fujifilm (1 mentions)
Las 3000 imaging system, by Fujifilm (1 mentions)
Histone h3, by Merck Group (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
4 protocols using amersham enhanced chemiluminescence reagent
1
Western Blot Protocol for Cellular Protein Analysis
2
SDS-PAGE analysis of GNPNAT1/GNA1 and AKT
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Cells were lysed with the same method of protein digestion as discussed earlier. Proteins (30 μg) were loaded onto 12.5% SDS-PAGE, separated, and then transferred to polyvinylidene fluoride membranes. The membranes were then blocked in 5% skim milk and incubated with primary antibodies (Abcam, Cambridge, UK) against GNPNAT1/GNA1, AKT, phosphor-Ser473-AKT (p-AKT), and β-actin. The blots were developed by chemiluminescence using Amersham enhanced chemiluminescence reagents (GE Healthcare, Little Chalfont, UK). For GNA1 analysis, three groups of sample were added, that is, 1 M pure ALB, 2 (2 M), and 4 (4 M) fold of PTX.
3
Western Blot Analysis of OSCC Proteins
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OSCC cells were lysed using a lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 1 mM PMSF, protease/phosphatase inhibitor cocktail) for the collection of total protein. The protein concentrations of samples were quantified using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific). Equal amounts of protein were loaded onto a sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, and transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) and subsequently incubated with primary antibodies overnight at 4°C. After washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The detected proteins were visualized using the Amersham enhanced chemiluminescence reagent (GE Healthcare, Little Chalfont, UK) and analyzed using Da Vinci software.
4
Western Blot Analysis of CACNA1G Protein
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Tissues and cells were lysed with 2× sample buffer (4% w/v sodium dodecyl sulfate [SDS], 20% glycerol, 200 mM dithiothreitol [DTT], 0.1 M Tris-HCl, pH 6.8, and 0.02% bromophenol blue), including protease and phosphatase inhibitors. Protein samples were quantified using a standard bicinchoninic acid (BCA) analysis and an equal amount of total protein was resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 1% skim milk and incubated overnight at 4°C with primary antibodies against CACNA1G, (ab134269; Abcam, Cambridge, UK; 1:2,000), β-actin (A2066; Sigma-Aldrich; 1:50,000), Histone H3 (9715S; Cell Signaling Technology, Beverly, MA, USA; 1:2,500), or acetylated Histone H3 (06-599; Millipore, Billerica, MA, USA; 1:2,500). After washing each membrane three times with 0.1% Tris-buffered saline (TBS-Tween), the blots were incubated with the peroxidase-conjugated secondary antibody for 2 h at 20-25°C. After washing the blots with 0.1% TBS-Tween, bands were detected using Amersham enhanced chemiluminescence reagent (GE Healthcare Life Science, Pittsburgh, PA, USA) and visualized using a LAS-3000 imaging system (Fuji Film, Tokyo, Japan). The band intensity was analyzed using the Multi Gauge v3.0 software (Fuji Film).
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