Normal human epidermal keratinocytes were seeded into 12-well plates and cultured as described above. Cells were collected and disrupted in lysis buffer (Cell Signaling Technology, Boston, MA, United States). After adding SDS sample buffer (Cell Signaling Technology), lysates were electrophoretically separated on a 12% polyacrylamide gel (ATTO Corp., Tokyo, Japan). Proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, United States). The membrane was blocked in 5% non-fat dry milk in Tris–buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 h at room temperature. After several washes with TBST, the membrane was incubated overnight at 4°C with primary mouse anti-human IL-36 beta antibody (R and D system; 1:1000), anti-human IL-36 gamma antibody (R and D system; 1:1000), anti-human p19 antibody (Proteintech, Tokyo; 1:1000), or anti-human tublin antibody (Proteintech; 1:1000). The membrane was washed several times in TBST followed by a 1-h incubation with horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Santa Cruz, Dallas, TX, United States).
Polyacrylamide gel
Manufactured by ATTO Corporation
Sourced in Japan
12% polyacrylamide gel is a laboratory equipment used for electrophoresis. It is a semisolid matrix made of polymerized acrylamide monomers at a concentration of 12%. This gel is used to separate and analyze macromolecules, such as proteins or nucleic acids, based on their size and charge.
Automatically generated - may contain errors
Lab products found in correlation
Lysis buffer, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated goat anti mouse igg secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Sds sample buffer, by Cell Signaling Technology (2 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (2 mentions)
Radioimmunoprecipitation assay buffer, by Fujifilm (2 mentions)
Nanodrop one spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Horseradish peroxidase hrp linked anti rabbit igg antibody, by GE Healthcare (1 mentions)
Anti rat igg antibody, by GE Healthcare (1 mentions)
Enhanced chemiluminescence plus western blotting detection system, by GE Healthcare (1 mentions)
Horseradish peroxidase hrp linked anti mouse igg antibody, by GE Healthcare (1 mentions)
Enhanced chemiluminescence plus detection system, by GE Healthcare (1 mentions)
4 protocols using polyacrylamide gel
1
Immunoblot Analysis of IL-36 Cytokines
2
Western Blot Analysis of IL-23p19 in A375 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A375 cells were seeded onto 6-well plates and cultured as described above. Cells were collected and disrupted in lysis buffer (Cell Signaling Technology, Boston, MA, USA). After adding SDS sample buffer (Cell Signaling Technology), lysates were electrophoretically separated on a 12% polyacrylamide gel (ATTO Corp., Tokyo, Japan). Proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.1% Tween-20 (TBST) for 1 h at room temperature. After several washes with TBST, the membrane was incubated overnight at 4 °C with primary mouse anti-human IL-23p19 antibody (Lifespan Bioscience, 1:1000) or mouse anti-human beta-actin antibody (Cell Signaling Technology, Tokyo, Japan; 1:1000). The membrane was washed several times in TBST followed by 1 h incubation with horseradish peroxidase–conjugated goat anti-mouse IgG secondary antibody (Santa Cruz, CA, USA).
3
Western Blot Analysis of Transcription Factor Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed to analyze TF expression in cultured cells. Whole-cell lysates were prepared using radioimmunoprecipitation assay buffer (Wako Pure Chemical Industries, Osaka, Japan) supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Total protein concentration was measured using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). Protein samples (35 μg) were separated on a 4%-20% polyacrylamide gel (ATTO Corporation, Tokyo, Japan) and transferred on to Immobilon-P membrane (Millipore, Billerica, MA, United States). As primary antibodies, anti-TF 1849 (generated by us) and a commercially available goat anti-human actin (C-11) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, United States), were used. A horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody and anti-rat IgG antibody (GE Healthcare, Little Chalfont, United Kingdom) were used as the secondary antibodies. Immunoreactive bands were visualized using the Enhanced Chemiluminescence Plus Western blotting detection system (GE Healthcare).
4
Folate Receptor Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Folate receptor expression in designated cells was examined by western blotting (WB). First, whole-cell lysate was prepared using radioimmunoprecipitation assay buffer (Wako) containing protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO, United States). Total protein concentration was determined using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, United States). Next, 50 μg of cell lysate protein samples were separated using 4%-20% polyacrylamide gel (ATTO Corporation, Tokyo, Japan) and transferred onto an Immobilon-P membrane (Millipore, Billerica, MA, United States). Mouse monoclonal antibody anti-FR of human origin (E-11) (Santa Cruz Biotechnology, Santa Cruz, CA, United States) (1:200 dilution) and goat polyclonal anti-human actin antibody (Santa Cruz Biotechnology) (1:500 dilution) were used as primary antibodies. Horseradish peroxidase (HRP)-linked anti-mouse IgG antibody (GE Healthcare, Little Chalfont, United Kingdom) (1:1000 dilution) and HRP-linked anti-goat IgG antibody (Santa Cruz Biotechnology) (1:1000 dilution) were used as secondary antibodies. The Enhanced Chemiluminescence Plus detection system (GE Healthcare) was used to visualize the immunoreactive bands.
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!