Western blot analysis was performed on 30 μg of whole cell protein from each lung graft after 4 hours EVLP as described previously. The following primary antibodies were used: anti-inhibitor of κB (IκB)-α/p-IκB-α (Novus Biologicals, LLC, Centennial, Colo) and anti-β-actin (MilliporeSigma). After incubation with horseradish-peroxidase conjugated goat anti-rabbit or anti-mouse secondary antibodies (Pierce Chemical, Rockford, Ill), the membranes were developed with the SuperSignal detection system (Pierce Chemical), and signals were detected by Imaging System (ChemiDoc, Bio-Rad, Hercules, Calif). Expression of the listed proteins was quantified by ImageJ (National Institutes of Health) and normalized with β-actin level.
Supersignal detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The SuperSignal detection system is a laboratory equipment product designed to enable sensitive and accurate detection of biological molecules. It provides a reliable platform for various analytical techniques without additional interpretation.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by Agilent Technologies (3 mentions)
X ray film, by GE Healthcare (3 mentions)
Bca protein assay kit, by Solarbio (2 mentions)
Chemidoc, by Bio-Rad (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Protease inhibitor mix, by Roche (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Horseradish peroxide hrp conjugated anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Skim milk powder, by Merck Group (1 mentions)
Protease inhibitor mixture, by Roche (1 mentions)
12 protocols using supersignal detection system
1
Protein Expression Analysis in Lung Grafts
2
Protein Expression Analysis in Liver Tissue and AML12 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen liver tissue and AML12 cells were homogenized in RIPA lysis buffer and fully dissociated. The mixture was centrifuged at 12000 rpm for 15 min. The supernatant was collected. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Solarbio). Samples were mixed with loading buffer and run on a 12% sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% skim milk for 1 h and then incubated at 4 °C overnight with primary antibodies for IRF-1, P38, p-P38, Caspase-3, LC3, P62 and GAPDH. After being washed with TBST and incubated with HRP-conjugated secondary antibody for 1 h at room temperature, membranes were washed and developed with the Super Signal detection system (Pierce Chemical) and exposed to film.
3
Western Blot Analysis of Endothelial Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Thirty micrograms of cytosolic protein was electrophoresed on 12% acrylamide sodium dodecyl sulfate gels and was transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). To block non-specific binding, 5% non-fat dry milk in PBS-Tween (0.1%) was added to the membrane for 1 h at room temperature. Membranes were washed in PBS-Tween then incubated overnight with mouse mAbs for eNOS, p-eNOS, AktT, p-AkT and GTPCH (dilution 1:1,000; Santa Cruz Biotechnology, Shanghai, China), and a rabbit anti-mouse polyclonal antibody for mouse β-actin (dilution 1:100; Kangwei Shiji Biotechnology, Peking, China) followed by incubation with a secondary antibody for 1 h. After repeat washings with PBS-Tween, membranes were developed using the SuperSignal detection system (Pierce Chemical, Rockford, IL, USA) and were exposed to film. Quantification of the western blot analyses for eNOS, p-eNOS, Akt, p-Akt and GTPCH was performed using gel analysis software.
4
Protein Expression Analysis in Liver Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen liver tissue and AML12 cells were homogenized in RIPA lysis buffer and fully dissociated. The mixture was centrifuged at 12000 rpm for 20 min. The supernatant was collected. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Solarbio). Samples were mixed with loading buffer and run on a 12% sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE). The proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% skim milk for 1 h and then incubated at 4℃ overnight with primary antibodies for IRF-1, LC3, Bcl-2, Beclin 1 and β-actin. After being washed with TBST and incubated with the HRP-conjugated secondary antibody for 1 h at room temperature, membranes were washed and developed with the Super Signal detection system (Pierce Chemical) and exposed to film. The experiments were repeated at least three times.
5
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted with 2X Laemmli buffer and analyzed by SDS-PAGE and Western blotting. Immunoreactive bands were detected by chemiluminescence using chemidoc (Bio-Rad Laboratories, Marnes la Coquette, France) with the SuperSignal detection system (Pierce Biotechnology Inc., Rochefield, IL, USA).
6
Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted with 2× Laemmli buffer and analyzed by SDS-PAGE and Western blotting. Immunoreactive bands were detected by chemiluminescence using chemidoc (Bio-Rad Laboratories, Marnes la Coquette, France) with the SuperSignal detection system (Pierce Biotechnology Inc., Rochefield, IL, USA).
7
Western Blot Analysis of Ovarian Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ovarian samples were lysed in WIP lysis solution (8553S, Cell Signaling Technologies, USA). Sample proteins were separated by electrophoresis by 7.5% SDS-PAGE and then transferred to PVDF (polyvinylidene fluoride) membranes (IPVH00010, Millipore, USA). The membranes were blocked with 5% nonfat-dry milk for 1 h at room temperature and incubated at 4 °C overnight with appropriate primary antibodies. The antibodies used were listed in Table S1 . After that, the membranes were washed thoroughly with tris buffered saline tween (TBST) and incubated with the secondary antibody (1:4,000, ZSGB-BIO, China) for 1 h at 37 °C and rinsed thoroughly with TBST. The membranes were visualized by the SuperSignal detection system (Prod 34080, Thermo, USA). To quantify the results of immunoblot, the image was quantified using Image J software.
8
HEK293T Cell Fractionation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For whole culture extracts, transfected HEK293T cells were lysed in Triton X-100 Lysis Buffer (150 mM NaCl, 50 mM Tris HCl, 1% Triton X-100, pH 8,0, protease inhibitor mix, Roche Applied Science, Mannheim, Germany). The NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Bonn, Germany) were further used to obtain nuclear and cytoplasmic fractions from transfected HEK293T cells. Protein concentrations were determined by Bradford protein assay, and the same amount of protein was loaded per lane for SDS-PAGE. Western blot analysis was conducted following standard protocols. HRP-conjugated secondary antibodies (Dako, Glostrup, Denmark) and the SuperSignal detection system (Thermo Scientific, Bonn, Germany) were used to visualize protein bands on X-ray films (GE Healthcare, Freiburg, Germany).
9
Quantification of ENO1 Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PDAC cells (1×107) from the various cell lines were harvested, lysed, resolved and transferred to nitrocellulose membranes, as previously described [40 (link)]. Membranes were incubated for 1 h at RT with anti-ENO1 72/1 mAb or rabbit polyclonal anti- β-Actin antibody (Sigma-Aldrich), at dilutions of 1:2000 in Tween-Tris-Buffered Saline (TTBS) and then probed with a horseradish peroxide (HRP)-conjugated anti-mouse IgG (Santa Cruz) or HRP-conjugated goat anti-rabbit Ig secondary antibody (Sigma-Aldrich) at dilutions of 1:2000. For Western blot analysis of ENO1 in PANC-1/P, PANC-1/M and HPDE cells, membranes were probed with in-house purified rabbit antiserum against ENO1 or with mouse antibody specific to β-Actin (Sigma St. Louis, MO, USA) as a protein loading control. Immunocomplexes were detected by probing with appropriate secondary antibodies conjugated with HRP (Jackson ImmunoResearch), and were visualized using the SuperSignal detection system (Thermo Fisher, Waltham, MA, USA).
10
Western Blot Analysis of Cerebral Cortex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot of brain tissue was performed as previously reported [23 (link)]. In brief, cerebral cortex either of WT or dKO mice were homogenized in HEPES-buffered sucrose (320 mM sucrose, 5 mM HEPES, pH 7.4) containing protease inhibitors. Equal amounts of 10 µg protein were separated by SDS-PAGE and subsequently blotted on nitrocellulose membranes according to standard protocols. Membranes were blocked with 5% skim milk powder (Sigma-Aldrich, Burlington, Massachusetts, US) in Tris-buffered saline with 0.1% TWEEN-20 (TBST). After overnight incubation with primary antibodies, the membranes were washed 3 times with 0.1% TBST and incubated with HRP-conjugated secondary antibodies (Dako, Agilent Technologies, Santa Clara, California, US) for 1 h. Membranes were then washed 3 times with 0.1% TBST and immunoreactivity was visualized on X-ray film (GE Healthcare, Chicago, Illinois, US) using the SuperSignal detection system (Thermo Scientific, Waltham, Massachusetts, US).
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!