HT-29 cells were propagated in RPMI-1640 with 10% FCS. HT-29 cells were exposed to L-mimosine (0-500 μmol/L) for 5 hours at 37°C. Cells were then washed with PBS. For gene expression experiments, total RNA was extracted with Trizol reagent (Life Technologies) and qPCR for selected genes was carried out as noted above. For protein analysis, protein was extracted with RIPA buffer, and concentrations determined using BCA assay (Pierce). Proteins were separated by 4-12% Tris-tricine (Invitrogen) gel electrophoresis. Western blot analysis was performed using anti HIF-1α (NB100-449, Novus) and anti LL-37 (NBP1-76864, Novus) rabbit polyconal antibodies. The secondary Ab was a horseradish (HRP) peroxidase-conjugated goat anti-rabbit (7074, Cell Signaling). Actin (pan-Actin rabbit monoclonal antibody, D18C11, Cell Signaling) was used as an internal control to confirm equal protein loading. Immunoreactive proteins were detected using the ECL-chemiluminescent system (EMD Millipore Immobilon).
Tris tricine
Manufactured by Thermo Fisher Scientific
Tris-tricine is a chemical buffer solution used in electrophoresis techniques, specifically for separating low-molecular-weight proteins. It provides a stable pH environment for the electrophoretic separation process.
Automatically generated - may contain errors
Lab products found in correlation
Nbp1 76864, by Novus Biologicals (2 mentions)
Horseradish peroxidase hrp conjugated goat anti rabbit, by Cell Signaling Technology (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Irdye 800 goat anti rabbit igg, by LI COR (1 mentions)
Irdye 680 goat anti mouse igg, by LI COR (1 mentions)
Tris glycine, by Bio-Rad (1 mentions)
Goat anti rabbit igg hrp, by Abcam (1 mentions)
Goat anti mouse igg hrp, by Abcam (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
3 protocols using tris tricine
1
Evaluating L-mimosine's Influence on HIF-1α and LL-37 Expression
2
Western Blot Analysis of Bacterial Proteins
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Western blot analysis was performed as described previously with minor changes (Beisel and Storz 2011 (link); Thomason et al. 2012 (link)). Samples were separated on a precasted 5%–20% Tris-Glycine (Bio-Rad) or 16% Tris-Tricine (Invitrogen) and transferred to a nitrocellulose membrane (Invitrogen). Membranes were blocked in 5% milk. To detect Lpp, the blocked membranes were probed with a 1:100,000 dilution of α-Lpp antibody (kindly provided by the laboratory of T. Silhavy) followed by incubation with a 1:20,000 dilution of HRP goat anti-rabbit IgG (Abcam) or a 1:10,000 dilution of IRDye800 goat anti-rabbit IgG (Licor). To detect GroEL, the membranes were incubated with a 1:20,000 dilution of α-GroEL mouse monoclonal (Abcam) followed by incubation with a 1:40,000 dilution of HRP goat anti-mouse IgG (Abcam). For both Lpp and GroEL, the membranes were developed using SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and exposed to KODAK Blue-XB film. To detect RpoA, the membranes were incubated with a 1:1000 dilution of α-RpoA mouse monoclonal antibody (Neoclone) followed by incubation with 1:10,000 IRDye680 goat anti-mouse IgG (Licor). Fluorescent antibodies were visualized on an Odyessy imager (Licor).
3
Evaluating L-mimosine's Influence on HIF-1α and LL-37 Expression
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