H-ferritin Western blotting was performed with rabbit anti-human H-ferritin antibody (Santa Cruz; sc-376594; 400ng/mL), followed by HRP-labeled anti-rabbit IgG antibody. To detect Pit-1, Pit2 and LAMP1 were used rabbit anti-human Pit1 antibody (Abcam; ab177147; 2000 ng/mL), rabbit anti-human Pit2 antibody (Proteintech; 12820–1-AP; 60 ng/mL), rabbit anti-human-LAMP1 antibody (Abcam; ab24170; 1000 ng/mL). Western blot analysis for ENPP2 was performed using anti-human ENPP2 (Thermo Fisher Scientific; PA5–12478; 4000 ng/mL). Sox9 Western blot was performed with rabbit anti-human Sox9 (Abcam; ab26414; 2000 ng/mL). Western blots were performed with: rabbit anti-human Sox9 (Abcam; ab26414; 2000 ng/mL) and rabbit anti-human RUNX2 (Proteintech; 20700-I-AP; 400 ng/mL); rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5–19810; 400 ng/mL); rabbit anti-human IL1-β (Invitrogen; 17h18l16; 400 ng/mL). Complexes of antigen-antibody were visualized with a horseradish peroxidase chemiluminescence detection system (Amersham Biosciences; RPN2109). Membranes were reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Ab177147
Manufactured by Abcam
Ab177147 is a rabbit monoclonal antibody. It is designed for use in immunohistochemistry applications.
Automatically generated - may contain errors
2 protocols using ab177147
1
Comprehensive Protein Detection Assay
2
Quantification of Duodenal Protein Expression
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Duodenal lysates were prepared as described in Coudert et al.(16 (link)). Samples were denatured at 75°C for 10 min before loading (80 μg of protein) for migration on 10 % SDS/PAGE gels and then transferred to nitrocellulose membranes. Membranes were blocked in 5 % fat-free milk/PBS, 0⋅1 % Tween for 1 h at room temperature. Membranes were incubated with antibodies against SLC20A1 (ab177147; Abcam®) 1:5000 for 3 h and against CALB1 (ab25085; Abcam®) 1:500 overnight. Membranes were then washed several times and incubated for 1 h with goat anti-rabbit secondary antibodies (A21076, AlexaFluor®; Life Technologies). After stripping, membranes were incubated with anti-vinculin antibody (V9131; Sigma) 1:40 000 for 3 h and then incubated with rabbit anti-mouse secondary antibodies (A21065, AlexaFluor®; Life Technologies). Bands were visualised by IR fluorescence using an Odyssey® Imaging System (LI-COR Inc. Biotechnology) and quantified by Odyssey IR imaging system software (Application software, version 1.2).
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