Immunoblot analysis was performed as described previously [29] (link). Proteins were resolved by SDS-10% polyacrylamide gel electrophoresis. The blots were first incubated with rabbit anti-RAGE(1∶2000), rabbit anti-Actin (I-19; 1∶2000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-Myc (9E10) (sc-40; 1∶1000, Santa Cruz Biotechnology, Inc.), rabbit anti-Cdx1 antibody, and then reacted with horseradish peroxidase-conjugated anti-rabbit IgG, anti-mouse IgG (Cell Signaling Tech., Beverly, MA), or anti-goat IgG (Santa Cruz Biotechnology, Inc.) (second antibody dilution, 1∶1000). All antibodies reactions were performed in Blocking one (Nacalai Tesque). Chemiluminescence signals were obtained from reaction with Chemi Lumi One Plus Reagent (Nacalai Tesque), and monitored by LAS4000 system (FUJI film, Tokyo, Japan). All images were obtained within 5-min in adequate mode.
Chemi lumi one plus reagent
Manufactured by Nacalai Tesque
Sourced in Japan
Chemi Lumi One Plus Reagent is a luminescent detection solution used for western blotting and other immunoassay techniques. It provides a sensitive and quantitative chemiluminescent signal for the detection of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000 system, by Fujifilm (3 mentions)
Anti phosphorylated stat3, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Mouse anti myc 9e10 sc 40, by Santa Cruz Biotechnology (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Phosphorylated ampk, by Cell Signaling Technology (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti phosphorylated nf κb p65, by Cell Signaling Technology (1 mentions)
Anti β actin, by Medical & Biological Laboratories (1 mentions)
Horseradish peroxidase conjugated anti mouse or anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti nf κb p65, by Cell Signaling Technology (1 mentions)
3 protocols using chemi lumi one plus reagent
1
Immunoblot Analysis of RAGE, Actin, Myc, and Cdx1
2
Quantification of Phosphorylated Proteins
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Western blot analysis was performed, as described previously [24 (link)]. Briefly, 50 µg total proteins were blotted to 10% SDS-PAGE and transferred to PVDF membrane and reacted with anti-phosphorylated-STAT3 (1:1000 dilution) or phosphorylated-AMPK (1:1000 dilution) (Cell Signaling Tech., Danvers, MA, USA) and anti-Actin (1:1000 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and then exposed to horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Chemiluminescence signals were obtained from reaction with Chemi Lumi One Plus Reagent (Nacalai Tesque), and data were obtained using an LAS4000 system (FUJI film, Tokyo, Japan).
3
Western Blot Analysis of NF-κB and STAT3
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Western blot analysis was performed using a modified method described previously [23 (link)]. Each antibody was used at the following dilutions:
anti-phosphorylated-NF-κB/p65 (product no. 3033), 1:1,000 dilution; anti-NF-κB/p65 (product no. 3034), 1:1,000 dilution; anti-phosphorylated-STAT3 (product no. 9131), 1:1,000 dilution;
anti-STAT3 (product no. 9139); Cell Signaling Technology, Danvers, MA, USA), 1:1,000 dilution; and anti-β-actin (code no. PM053; Medical & Biological Laboratories Co., Ltd.), 1:1,000
dilution. Then, the membrane was exposed to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (product no. 7074; Cell Signaling Technology). Chemiluminescence signals were
obtained from reactions with Chemi Lumi One Plus Reagent (Nacalai Tesque), and data were obtained using an LAS4000 system (Fujifilm, Tokyo, Japan). β-actin was used as an internal control to
normalize differences in the amount of protein appliqued.
anti-phosphorylated-NF-κB/p65 (product no. 3033), 1:1,000 dilution; anti-NF-κB/p65 (product no. 3034), 1:1,000 dilution; anti-phosphorylated-STAT3 (product no. 9131), 1:1,000 dilution;
anti-STAT3 (product no. 9139); Cell Signaling Technology, Danvers, MA, USA), 1:1,000 dilution; and anti-β-actin (code no. PM053; Medical & Biological Laboratories Co., Ltd.), 1:1,000
dilution. Then, the membrane was exposed to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (product no. 7074; Cell Signaling Technology). Chemiluminescence signals were
obtained from reactions with Chemi Lumi One Plus Reagent (Nacalai Tesque), and data were obtained using an LAS4000 system (Fujifilm, Tokyo, Japan). β-actin was used as an internal control to
normalize differences in the amount of protein appliqued.
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