Total cell extracts were prepared by lysing the cells for 15 min in NP-40 buffer (50 mM Tris–HCl, pH 7.5, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM sodium pyrophosphate, 1 mM PMSF, 0.1% Nonident P-40) followed by centrifugation for 20 min at 14,000 × g. Cleared lysates were analyzed directly by SDS-PAGE and western blotting. Proteins were visualized using Enhanced Chemiluminescent solution (Thermo Fisher) and a FUSION Vilber imager. Anti-HIF-1α (#2185), anti-TPI (ab96696), anti-PGK1 (ab38007), and anti-PDK1 (EPR19573, ab207450) were obtained from Abcam. β-Actin was detected as loading control (8H10D10, Cell Signaling Technology). Raw images of the complete blots for the experiments shown are provided as Supplementary information.
Enhanced chemiluminescent solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Enhanced Chemiluminescent solution is a laboratory reagent designed to facilitate chemiluminescent detection and quantification of proteins in Western blot analysis. It provides a sensitive and reliable method for visualizing target proteins on membranes.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
Anti akt, by Santa Cruz Biotechnology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Anti actin 8h10d10, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
Pierce lane marker reducing sample buffer, by Thermo Fisher Scientific (1 mentions)
Imagequant 1d software, by GE Healthcare (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
5 protocols using enhanced chemiluminescent solution
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Skin Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
C57BL6/N dorsal skin tissues were homogenized and lysed in 1% RIPA buffer containing protease and phosphatase inhibitors (Roche, Mannheim, Germany), and total proteins were separated on 10% SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes, and the membranes were blocked with 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20. The membranes were immunoblotted with primary antibodies, including anti-β-catenin, anti-phospo-Akt, anti-Akt, and anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (Stressgen, San Diego, CA, USA). The blots were developed using an enhanced chemiluminescent solution (Thermo, Rockford, IL, USA).
3
Quantification of HIF-1α in NK Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell extracts were prepared by resuspending 3 × 106 NK cells in 100 μL NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM sodium pyrophosphate, 1 mM PMSF, 0.1% Nonident P-40). Fifteen minutes of cell lysis on ice was followed by centrifugation for 20 min at 14,000 × g. Cleared lysates were analyzed directly by SDS-PAGE and Western Blotting. Briefly, equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes (Thermo Fisher), blocked in 5% dry milk powder dissolved in 1×PBS-T, and then probed with primary antibody and HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were visualized using Enhanced Chemiluminescent solution (Thermo Fisher) and FUSION Vilber imager (Eberhardzell, Germany). The intensity of signals was quantified by densitometric analysis using the image analysis software ImageJ (Version 1.51j8). The value for HIF-1α was normalized to that for β-Actin. Anti-HIF-1α (# 2185) was obtained from Abcam (Cambridge, UK) and Anti-Actin (8H10D10) from Cell Signaling Technology (Frankfurt am Main, Germany). Representative experiments out of three performed are shown.
4
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 24 h treatment with DAU or vehicle, cells were collected and lysed in 200 μL of IP buffer with protease and phosphatase inhibitor cocktail on ice for 20 min and centrifuged at 14,000g at 4°C for 20 min. Supernatants were used for protein content determination and SDS-PAGE separation. The total protein content of each sample was determined with the Pierce BCA protein assay kit. Before loading onto the SDS-PAGE gel, samples were mixed with Pierce Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific, Rockford, IL, USA) and denatured (boiled for 10 min). SDS-PAGE (10–12%) gels were used to separate target proteins and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore Ltd., Merck KGaA, Darmstadt, GER). Membranes were blocked with nonfat milk powder dissolved in TBS-Tween 20 buffer for 2 h and then incubated with primary antibody (dilutions of the antibodies are listed in Table 1 ) at 4°C overnight. The membranes were washed and incubated with anti-mouse, anti-rabbit, or anti-goat IgG conjugated to horseradish peroxidase (HRPs) (1 : 3000) at room temperature (RT) for 1 h before development. Enhanced chemiluminescent solution (Thermo Fisher Scientific, Rockford, IL, USA) was applied for development. The densitometry of the blots was quantified by ImageQuant 1D software (GE. Healthcare, Pittsburgh, PA, USA).
5
Western Blot Analysis of PPAR-γ and C/EBP-α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (Cell Signaling, USA) containing 1% protease inhibitor cocktail, 0.5 mM DTT, and 1 mM PMSF. Supernatants were attained through centrifugation for 15 min at 12,000 × g. The protein assay was conducted using Bradford protein assay kits (Bio-Rad, USA). The mixture of total proteins (20 μg) and sample loading buffer (Biosaesang Co., Korea) was boiled at 100°C for 10 min for Western blot analysis. The denatured proteins were separated through 10% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). After blocking in TBS-T with 5% non-fat dry milk, the membranes were incubated overnight at 4°C with 1:1000 diluted peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP) α primary antibodies (Cell Signaling, Beverly, USA). After incubation with 1:1000 diluted horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (Santa Cruz Biotechnology, USA) for 1 h at RT, immunoreactive proteins were developed by an enhanced chemiluminescent solution (Thermo Scientific, USA). The density values for the protein bands were expressed as a percentage of the control after normalization to β-actin.[14 (link)]
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!