A total of 1x106 293T cells were transduced with 1x106 CFU of each viral construct in the presence of 1ug/ml doxycyline. After 48 hours, cells were harvested and lysed in RIPA buffer (Biorad) for Western blot. Proteins were denatured in 2% SDS with 1% DTT before loading on a 4–15% gradient Tris Glycine-SDS poly- acrylamide gels (Bio-Rad), electrophoresed, and blotted onto PVDF membrane (Pierce). The membrane was blocked using 5% (w/v) dry milk and then probed with Mouse anti-EBV LMP-1 antibody (Santa Cruz Biotechnology), followed by incubation with Peroxidase-Conjugated Donkey Anti-mouse IgG (Jackson Immunoresearch). The protein band was developed onto X-ray film using ECL detection reagent (Amersham).
Tris glycine sds poly acrylamide gels
Manufactured by Bio-Rad
Tris Glycine-SDS poly-acrylamide gels are used for the separation and analysis of proteins based on their molecular weight. They are a type of electrophoresis gel composed of a polyacrylamide matrix and a Tris-Glycine-SDS buffer system.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Bio-Rad (1 mentions)
Ecl detection reagent, by Cytiva (1 mentions)
Peroxidase conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Protease and phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Hybond c extra, by GE Healthcare (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
100 mm petri dishes, by BD (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
G9545, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
3 protocols using tris glycine sds poly acrylamide gels
1
Quantitative EBV LMP-1 Expression Analysis
2
Western Blot Analysis of Protein Expression
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Cells were seeded in 100-mm Petri dishes (Falcon) and treated 24 h after as indicated. In mammalian protein extraction reagent (Pierce) complete with protease and phosphatase inhibitor cocktail (Sigma), cells were scraped and centrifugated to remove cellular debris. Protein concentrations of whole cell lysates were determined using the bicinchoninic acid protein assay (Pierce); samples were diluted in 5 mM dithiothreitol and Laemmli buffer (final 1×) and heated at 95°C for 5 min. At least 10 μg of total proteins were separated on 7.5% or 4–15% gradient Tris–glycine SDS-polyacrylamide gels (Bio-Rad), and then transferred onto PVDF membranes (Hybond-C Extra, GE Healthcare Life Sciences). Membranes were blocked in 2% BSA (Sigma) in 1× PBS for 1 h at RT. Primary antibodies (Supplementary Table S2 ) diluted in 2% BSA were incubated with membranes overnight at 4°C. Membranes were washed three times with 1× PBS–0.1% Tween™ 20 (Sigma) and bound antibodies were detected using peroxidase-conjugated secondary antibodies (Santa Cruz), followed by enhanced chemiluminescence (Pierce). Signal was detected using ChemiDoc™ MP Imaging System (Bio-Rad). GAPDH was used to normalize results using the ImageLab 6 Software (Bio-Rad).
3
Denaturing SDS-PAGE Gel Preparation and Analysis
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For denaturing SDS-PAGE gels, fibroblasts were grown to confluency, washed twice in ice-cold PBS, then extracted in radioimmunoprecipitation assay buffer (10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, 1 mM PMSF, and protease inhibitor cocktail [Roche]) with gentle sonication, and then incubated on ice for 30 min before centrifugation at 20,000 g for 20 min at 4°C. Protein concentration was determined from the supernatant using the Pierce BCA Protein Assay Kit. SDS-PAGE was performed on 10–30 µg of cell lysate per sample on Tris-glycine-SDS polyacrylamide gels (Bio-Rad) and then transferred to Amersham polyvinylidene fluoride using standard techniques.
Antibodies were specific to the N-terminus of EXOC2 (1:1,000; ab140620; Abcam) and GAPDH (1:10,000; G9545; Sigma Aldrich). GAPDH was used as a loading control. Primary antibodies were detected with appropriate anti-mouse or anti-rabbit HRP-conjugated antibodies (GE Healthcare) using enhanced chemiluminescence reagents (GE Healthcare) and Amersham Hyperfilm. Protein band intensities were quantified using ImageJ software, and band intensity determined in the linear range was normalized to band intensity of GAPDH.
Antibodies were specific to the N-terminus of EXOC2 (1:1,000; ab140620; Abcam) and GAPDH (1:10,000; G9545; Sigma Aldrich). GAPDH was used as a loading control. Primary antibodies were detected with appropriate anti-mouse or anti-rabbit HRP-conjugated antibodies (GE Healthcare) using enhanced chemiluminescence reagents (GE Healthcare) and Amersham Hyperfilm. Protein band intensities were quantified using ImageJ software, and band intensity determined in the linear range was normalized to band intensity of GAPDH.
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