SDS-PAGE and western blot investigations were performed by previously developed protocol with slight modifications [19 (link), 20 (link)]. Briefly, after 24 h, and 48 h of treatments with NVD at vital doses, HCT116 and HT29 cells were lysed in DMEM augmented with freshly added protease and phosphatase inhibitor cocktail 1:100 (Santa Cruz, CA) and protein concentration were estimated by Bradford assay [21 ]. For western blotting 8–12% polyacrylamide gels were used to resolve 40 μM of protein, transferred on to a nitrocellulose membrane, probed with appropriate monoclonal primary antibodies, and detected by super signal west Pico, Dura or Femto Chemiluminescence Reagent (Thermo Scientific, USA). Quantification of protein bands was done through measuring band density using Image J software. The densities of the bands (normalized to actin) relative to that of the untreated control (designated as 1.00) were presented as mean ± SEM of three separate experiments.
Femto chemiluminescence reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Femto Chemiluminescence Reagent is a highly sensitive, ultra-low-light detection solution designed for quantitative Western blotting applications. It enables the detection of low-abundance proteins with high signal-to-noise ratios.
Automatically generated - may contain errors
Lab products found in correlation
Protease and phosphatase inhibitor cocktail, by Santa Cruz Biotechnology (2 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
Phenylmethylsulphonyl fluoride, by Merck Group (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Imagequant las 4000 chemiluminescent image analyzer, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
5 protocols using femto chemiluminescence reagent
1
SDS-PAGE and Western Blot Analysis
2
Evaluation of TAX Treatment in Colon Cancer
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SDS-PAGE and western blot investigations were executed by earlier described protocol with minor amendments [21 (link)]. Subsequent to 24 h and 48 h of administration with TAX at necessary amounts, HCT116 and HT29 cells lyses was carried out in RIPA buffer augmented with freshly added protease and phosphatase inhibitor cocktail 1:100 (Santa Cruz, CA) and concentration of protein was anticipated by Bradford assay [22 ]. To resolve 40-60 μg of protein, 8–12% poly acrylamide gels were used in immune-blotting. Transferred on to a nitrocellulose membrane, with candidate monoclonal primary antibodies, and perceived by super signal west Pico, Dura or Femto Chemiluminescence Reagent (Thermo scientific, USA). Protein bands were quantified by measuring band density using Image J software. The densities of the bands (normalized to actin) relative to that of the untreated control (designated as 1.00) were presented as mean ± SEM of three individual experiments.
3
Protein quantification and analysis by SDS-PAGE and Western blot
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The previously developed procedures with slight modifications were used to perform SDS-PAGE and western blot investigations. The protein concentration was estimated by Bradford assay. For western blotting 8–12% polyacrylamide gels were used to resolve 40 μM of protein, transferred on to a nitrocellulose membrane, probed with appropriate monoclonal primary antibodies, and detected by super signal west Pico, Dura or Femto Chemiluminescence Reagent (Thermo Scientific, USA). Quantification of protein bands was done through measuring band density using Image J software. The densities of the bands (normalised to actin) relative to that of the untreated control (designated as 1.00) were presented as mean ± SEM of three separate experiments.
4
Protein Extraction and Western Blot Analysis of Melanoma Xenografts
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Melanoma xenografts were excised and quickly snap frozen. Tissue lysates were prepared in Kontes tubes with disposable pestles using RIPA Buffer (Cell Signaling Technology) supplemented with phenylmethylsulphonyl fluoride (Sigma), and protease and phosphatase inhibitor cocktail (Roche). The bicinchoninic acid protein assay (Thermo) was used to quantify protein concentrations. Equal amounts of protein (10–20 μg) were separated on polyacrylamide gels and transferred to polyvinylidene difluoride membranes (BioRad). Membranes were blocked for 1 h at room temperature with 5% milk in TBS supplemented with 0.1% Tween20 (TBST) and subsequently incubated with primary antibodies overnight at 4 °C. After incubating with horseradish peroxidase conjugated secondary antibodies (Cell Signaling Technology), membranes were developed using SuperSignal West Pico or Femto chemiluminescence reagents (Thermo). Antibodies against pERK (p44/42 MAPK; Thr202/Tyr204; D13.14.4E) and ERK (p44/42 MAPK;137F5) were obtained from Cell Signaling Technologies. Antibodies against ATPA1A1 (C464.6), BCL2 (C-2) and Tubulin (TU-02) were obtained from Santa Cruz and used for western blot analyses. Uncropped scans of the main blots appear in Supplementary Fig. 8 .
5
Western Blot Protein Detection Protocol
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