Wild-type cells, talin B-null cells, and transformants expressing each GFP-protein were individually lysed in SDS buffer (50 mM Tris-Cl (pH 6.8), 2% SDS) containing protease inhibitor cocktail (Nacalai) and were heated at 95°C for 5 min. The lysates were then subjected to SDS-PAGE and were transferred to PVDF membranes, which were then incubated with a mouse anti-GFP antibody (Santa Cruz; sc-9996), a mouse anti-actin antibody (Millipore; MAB1501), or a rabbit anti-talin B antiserum [12 (link)]. The immune complexes were detected using HRP-conjugated anti-mouse or anti-rabbit secondary antibodies and Chemi-Lumi One Super reagent (Nacalai).
Chemi lumi one super reagent
Manufactured by Nacalai Tesque
Sourced in Japan
Chemi-Lumi One Super reagent is a luminescent substrate for the detection of horseradish peroxidase (HRP) conjugates in Western blotting and ELISA applications. The reagent generates a chemiluminescent signal that can be detected using a luminometer or X-ray film.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Nacalai Tesque (3 mentions)
Imagequant las 4000 system, by GE Healthcare (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Mab1501, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Ripa buffer, by Nacalai Tesque (1 mentions)
Bicinchoninic acid protein assay, by Thermo Fisher Scientific (1 mentions)
6 protocols using chemi lumi one super reagent
1
Talin B-null Cells Protein Analysis
2
Transglutaminase-Catalyzed Biotin Incorporation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant proteins were incubated with TG in 50 mM Tris-HCl, pH 8.5, containing 10 mM CaCl2, 10 mM DTT, and 500 μM biotin pentylamine at 37°C for 1 h. Following the reaction, the aliquots were subjected to SDS-PAGE and electroblotted on a polyvinylidene difluoride membrane. After blocking with 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 5% dry milk, the membrane was incubated at room temperature for 1 h with the horseradish peroxidase-conjugated streptavidin diluted 1:1,000 with blocking buffer, followed by development with Chemi-Lumi One-Super reagent (Nacalai Tesque).
3
Embryonic Epidermis Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The epidermis of embryos at embryonic day 18.5 (E18.5) were obtained by heat separation, as described previously37 (link). In brief, dorsal skin was incubated in PBS at 65 °C for 10 min and placed on ice for 1 min. The epidermis was isolated from the dermis using tweezers. Cultured cells were harvested using scrapers. Epidermis samples or cell samples were lysed in SDS buffer (50 mM Tris-HCl [pH 6.8], 2% SDS) containing a protease inhibitor cocktail (Nacalai Tesque) and subjected to sonication. Total lysates were separated on SDS-PAGE and proteins were transferred from polyacrylamide gels to polyvinylidene difluoride membranes (Millipore). Membranes were blocked with 3% skim milk in tris-bufferd saline (TBS) containing 0.1% Tween-20 (TBS-T) and incubated with primary antibodies. After washes in TBS-T, membranes were incubated with horseradish peroxidase-labeled secondary antibodies and visualized with ChemiLumi One Super reagent (Nacalai Tesque).
4
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and tissues were lysed in SDS buffer (50 mM Tris-Cl [pH 6.8] and 2% SDS) and RIPA buffer (Nacalai), respectively, followed by sonication. The lysates were then subjected to SDS-PAGE and were transferred to PVDF membranes, which were then incubated with a primary antibody. The immune complexes were detected using an HRP-conjugated secondary antibody and Chemi-Lumi One Super reagent (Nacalai). Original immunoblots are presented in Supplementary Figs. S11 –S23 .
5
Flavopiridol Modulates Protein Expression in CCA Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KKU-055 and KKU-213 CCA cells were treated with flavopiridol at different concentrations and times. Cells were lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM NaF, 1 mM Na3VO4) containing protease inhibitor cocktail (Nacalai Tesque, Tokyo, Japan). Protein amounts were determined by the bicinchoninic acid (BCA) protein assay (Thermo Science, Rockford, IL). Ten micrograms of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a PVDF membrane (GE Healthcare Japan, Tokyo, Japan), which was probed with primary antibodies. Horseradish peroxidase conjugated secondary antibodies were further incubated. Proteins were detected using Chemi-Lumi One Super reagents (Nacalai Tesque, Kyoto, Japan) and visualized with an ImageQuant Las4000 system (GE Healthcare Japan). β-actin was used as the internal control.
6
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KKU-213 cells were treated with CMA3 at indicated concentrations and times. Protein lysate was prepared as described elsewhere (29 (link)). Protein concentrations were determined by the bicinchoninic acid protein assay (Thermo Fisher Scientific, Inc.). A total of 20 µg of protein was separated by SDS-PAGE (10-15%) and transferred to a PVDF membrane (GE Healthcare Japan). The membrane was blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) at room temperature for 30 min and was incubated with specific primary antibodies (1:1,000) at 4°C overnight. Membranes were washed with TBST 3 times prior to the incubation with the corresponding HRP-conjugated secondary antibody (1:2,000) for 1 h at room temperature. Signals were detected using Chemi-Lumi One Super reagents (Nacalai Tesque, Inc.) and visualized by ImageQuant LAS 4000 system (GE Healthcare Japan). The quantitative analyses of blots were quantified by Image J (30 (link)). Hsc70 was used as an internal control.
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!