The DOPA staining was carried out as described previously (Nishio et al., 2016 (link)). B16F1 melanoma cells were treated with or without salicylamide (250-1,000 µM) for 3 days. After treatment, the cells were collected by trypsinization and lysed in 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin (but without mercaptoethanol or heating). After electrophoresis, SDS-PAGE gels were stained with 0.5 mg/mL L-DOPA for 2 h at 37°C. Gel images were collected with WSE-6100 LuminoGraph 1 (ATTO Corporation, Tokyo, Japan), and densitometric analysis was carried out using CS analyzer software (ATTO Corporation).
Cs analyzer software
Manufactured by ATTO Corporation
Sourced in Japan
The CS analyzer software is a data processing tool designed to analyze the results of carbon-sulfur (CS) analysis. It provides a platform for users to import, review, and interpret the data obtained from CS analysis instruments. The software's core function is to facilitate the presentation and interpretation of CS analysis data, allowing users to generate reports and compare results.
Automatically generated - may contain errors
Lab products found in correlation
X ray film, by Kodak (2 mentions)
Hsp70, by Abcam (1 mentions)
Ecl western blotting detection reagent, by Cytiva (1 mentions)
Imobiron, by Merck Group (1 mentions)
Protran ba85, by GE Healthcare (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel, by Bio-Rad (1 mentions)
Pierce g2 fast blotter, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
6 protocols using cs analyzer software
1
Melanoma DOPA Staining Protocol
2
Quantifying HSP70 Expression in Esophageal Tissue
3
Western Blot Analysis of HIF-1α
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples (5 μg) of the cell lysates were separated on a 15% (w/v) polyacrylamide gel 30 . Proteins were blotted onto a nitrocellulose membrane (Protran BA85, GE Healthcare UK) in a semidry blotting system (NA‐1513, Nihon Eidoh, Japan) 31 . Nitrocellulose membranes were blocked with 1% (w/v) BSA. Blocked membranes were incubated with anti‐HIF‐1α antibody for HIF‐1α (× 1000, Bethyl Laboratories, Montgomery, TX, USA), then with horseradish peroxidase (HRP)‐conjugated anti‐rabbit IgG goat antibody (Biosource, Camarillo, CA, USA). The blots were subsequently developed on a chemiluminescent detection system 32 or Imobiron (Merck Millipore, Billerica, MA, USA) using LuminoGraph (Atto, Amherst, NY, USA). The bands were densitometrically analyzed using cs analyzer software (Atto corp., Tokyo, Japan).
4
Western Blot Analysis of Insulin Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Insulin-resistant HepG2 cells were exposed to a range of concentrations of 1 –3 or rosiglitazone (10 μM) in 24-well plates for 24 h followed by stimulation with 100 nM insulin diluted in SFMEM for 30 min. Cells were then washed twice with ice cold PBS, collected, and lysed with sample buffer. Protein was quantified by the modified Bradford protein assay kit using bovine serum albumin (BSA) as a standard, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and then transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Burlington, MA, USA) using the Pierce G2 Fast Blotter (Thermo Fisher Scientific Inc, USA) at 100 V for 60 min in a standard semi-dry system. Membranes were blocked in blocking buffer and incubated overnight with primary antibodies at 4 °C on a shaker, followed by incubation with the appropriate secondary antibodies for 2 h at room temperature. The membranes were washed three times with tris-buffered saline with tween (TBST) and the protein bands were visualized with the Supersignal West Pico Chemiluminescence Substrate (Pierce, Rockford, IL, USA) on X-ray films (Kodak, Rochester, NY, USA) and quantified using CS analyzer software (Atto Corp., Tokyo, Japan). Detailed laboratory procedures and sample buffer and antibody solution compositions are reported in our previous paper [59 (link)].
5
Insulin Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In
brief, insulin-resistant HepG2 cells were treated with rubrofusarin
or rosiglitazone for 24 h and stimulated with 100 nM insulin for 30
min. Cells were then washed with ice-cold PBS, harvested, and lysed
with a sample buffer to extract protein. Then, aliquots of protein
were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred onto a nitrocellulose membrane, followed
by blocking the membrane with 10% nonfat milk (w/v) in TBST. After
that, the membrane was incubated overnight in primary antibodies and
in secondary antibody for 1 h and visualized with the Supersignal
West Pico chemiluminescence substrate (Pierce, Rockford, IL) on X-ray
films (Kodak, Rochester, NY) and quantified using CS analyzer software
(Atto Corp., Tokyo, Japan).
brief, insulin-resistant HepG2 cells were treated with rubrofusarin
or rosiglitazone for 24 h and stimulated with 100 nM insulin for 30
min. Cells were then washed with ice-cold PBS, harvested, and lysed
with a sample buffer to extract protein. Then, aliquots of protein
were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred onto a nitrocellulose membrane, followed
by blocking the membrane with 10% nonfat milk (w/v) in TBST. After
that, the membrane was incubated overnight in primary antibodies and
in secondary antibody for 1 h and visualized with the Supersignal
West Pico chemiluminescence substrate (Pierce, Rockford, IL) on X-ray
films (Kodak, Rochester, NY) and quantified using CS analyzer software
(Atto Corp., Tokyo, Japan).
6
Western Blot Analysis of Insulin-Resistant HepG2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Standard protocol was followed to prepare lysates of insulin‐resistant HepG2 cells using sample buffer and PMSF. Fifty micrograms of protein, once quantified by modified Bradford protein assay kit, was separated using a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio‐Rad, Hercules, CA). The polyvinylidene difluoride (PVDF) membranes were incubated overnight on a shaker at 4°C with primary antibody prepared in 5% skim milk and visualized on X‐ray film after incubating PVDF membranes with secondary antibody for 2 hr at room temperature. Band intensities were quantitated using CS analyzer software (Atto Corp., Tokyo, Japan).
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!