The total protein from each caecal mucosa sample was extracted with RIPA Lysis Buffer (cat.SN338, Sunshine Biotechnology Co., China). The concentration of the total protein was measured by the Pierce™ BCA Protein assay kit (cat.23225, Themo Fisher, USA). The detection metod of cadidate protein was indicated in the previous study [19 (link)]. The primary antibodies used in the current study included TLR4 (sc-293,072, Santa Cruz, USA), GPR41 (ab103718, Abcam, USA), GPR43 (ab131003, Abcam, USA), NF-κB (An365, Beyotime, China), p38 (ab31828, Abcam, USA), ERK1/2 (ab17942, Abcam, USA), β-tubulin (KC-5 T01, Kang Chen Bio-tech, China), and GAPDH (AP0066, Bioworld, USA). The expression level of each candidate protein is shown as the fold change of the average value between the LC and HC groups.
Ap0066
Manufactured by Bioworld Technology
Sourced in United States
The AP0066 is a laboratory centrifuge designed for general-purpose sample preparation and separation. It features a fixed-angle rotor that can accommodate various sample tubes or microplates. The centrifuge operates at variable speeds to facilitate efficient separation of samples based on their density and composition.
Automatically generated - may contain errors
Lab products found in correlation
Ab6728, by Abcam (2 mentions)
Ab31828, by Abcam (1 mentions)
Ab17942, by Abcam (1 mentions)
Ab131003, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
An365, by Beyotime (1 mentions)
Sc 293072, by Santa Cruz Biotechnology (1 mentions)
Ecl plus, by Thermo Fisher Scientific (1 mentions)
Zb 2301, by ZSGB-BIO (1 mentions)
Ripa buffer, by Aidlab (1 mentions)
Ta334206, by OriGene (1 mentions)
Odyssey sa, by LI COR (1 mentions)
Df12140, by Affinity Biosciences (1 mentions)
Ab1791, by Abcam (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ecl system, by GE Healthcare (1 mentions)
Nbp1 54467ss, by Novus Biologicals (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
7 protocols using ap0066
1
Caecal Mucosal Protein Profiling
2
Western Blot Analysis of Tissue Proteins
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Total tissue proteins were extracted with RIPA buffer (Aidlab Biotechnologies, Beijing, China). Tissue lysates were centrifuged at 14,000 rpm for 20 min at 4°C. Supernatant was collected and protein was quantified with a BCA reagent kit. Lysates were boiled at 100°C for 5 min and 40 g of total protein was separated by 4–12% SDS-PAGE at 110 V for approximately 2 h and transferred to polyvinylidene difluoride (PVDF) membranes at 70 V for 110 min. The PVDF membranes were blocked with 5% nonfat dry milk in PBST for 1 h on a table concentrator and then incubated with rabbit polyclonal anti—claudin-11 antibody (TA334203, 1:500, Origene, Rockville, USA), rabbit polyclonal anti—claudin-23 antibody (TA334206, 1:500, Origene, Rockville, USA), and rabbit polyclonal anti- GAPDH antibody (AP0066, 1:10000, Bioworld, Minnesota, USA) in 2.5% nonfat dry milk in PBST at 4°C overnight. The membranes were then incubated with secondary anti-IgG antibody (Zb2301, 1:10000, ZSGB-Bio, Beijing, China) for 1–2 h at room temperature. Chemiluminescence reagent ECL Plus (Thermo Fisher Scientific, Massachusetts, USA) was used to visualize the bands and the results were analyzed by Image J software.
3
Western Blot Analysis of Xenobiotic Enzymes
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Standard procedures performed western blotting. CYP1A1 (BS6575, Bioworld, USA), CYP1B1 (DF6399, Affinity Biosciences, USA), UGT1A (4371S, Cell Signaling Technology, Inc., USA), UGT2B (Ab113433, Abcam, UK), UGT2B7 (DF12140, Affinity Biosciences, USA) and GAPDH (AP0066, Bioworld, USA) primary antibodies were used individually for immunodetection. The intensities of the bands were quantified using Odyssey Sa (LICOR, USA).
4
Western Blot Analysis of KCNK1 and Reference Proteins
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RIPA was used to lyse cells or tissues to extract proteins. 20–50 μg of Protein was separated with 10% SDS‐PAGE gel, transferred onto a PVDF membrane, and incubated with antibody against KCNK1 (1:1000 arb390214 biorbyt) or GAPDH (1:5000 AP0066 bioworld) and H3 (1:2000 ab1791 abcam). Incubated in peroxidase‐conjugated goat anti‐rabbit IgG secondary antibody (1:1000; VA001, Vicmed) and visualized using chemiluminescence detection (72,091, Sigma‐Aldrich).
5
Western Blot Analysis of CTNNB1
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Total protein was extracted from cells using a RIPA buffer (Beyotime) supplemented with a protease inhibitor Cocktail (BioDee) (100:1). Protein was separated on 8% SDS-PAGE gels (GenScript). The proteins were transferred to PVDF membrane, and then blocked with 5% non-fat milk for 1 h. The membranes were incubated with the primary antibodies CTNNB1 (1:1000, NBP1-54467SS, Novus) and GAPDH (1:5000, AP0066, Bio-world) for 1h and washed with PBST (Solarbio) three times (5 min/time). Then the membranes were incubated with anti-mouse (1:3000, ab6728, Abcam) or anti-rabbit (1:3000, 7074P2, CST) secondary antibody conjugated with HRP for 1 h at room temperature and washed three times using PBST. Protein bands were visualized using enhanced chemiluminescence (ECL) system (GE Healthcare, USA) and quantified with ImageJ Software, as compared to GAPDH.
6
Western Blot Analysis of GIT1 Protein
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Western blotting was performed with standard procedures. Protein concentrations were detected using a Bio‐Rad protein assay kit. A 50‐μg sample of protein extract was separated by 10% SDS‐PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were then blocked in 5% non‐fat dry milk and incubated with the antibodies against GIT1 (1:1000, Abcam, ab153958) and GAPDH (1:5000, Bioworld, #AP0066). The membranes were incubated with a horseradish peroxidase‐conjugated secondary antibody for 1 hour at room temperature, and the protein bands were visualized using an electrochemiluminescence (ECL) chromogenic substrate and measured using densitometry (Quantity One software, Bio‐Rad).
7
Western Blot Analysis of Liver Proteins
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Western blot analysis was conducted essentially as described (Xu et al., 2015) . Briefly, protein was isolated from liver tissue homogenized in RIPA lysis buffer (Beyotime, Shanghai, China). Equal amounts of protein were separated on 10% SDS polyacrylamide gels to resolve SREBP1 and SCD1. Proteins were transferred onto nitrocellulose membranes (Millipore, Billerica, MA), which were incubated with primary antibodies against either SCD1 (Goat polyclonal antibody, sc-23016, Santa Cruz Biotechnology, Santa Cruz, CA; diluted to 1:200) or SREBP1 (Mouse monoclonal antibody, ab3259, Abcam, Cambridge, UK; diluted to 1:1,000). After extensive washing, blots were incubated with horseradish peroxidase-coupled secondary antibodies. We used affinity-purified rabbit anti-goat IgG antibodies (E030130-01, Earth Ox, San Francisco, CA; diluted to 1:10,000) to detect SCD1 and rabbit antimouse antibodies (ab6728, Abcam; diluted to 1:5000) to detect SREBP1. Differences in protein transfer efficiency between blots were normalized based on quantification of GAPDH, which was detected with rabbit polyclonal antibodies (AP0066, Bioworld, St. Louis Park, MN; diluted to 1:10,000). Intensities of bands from target genes were quantified with the Quantity One 1-D analysis software (Bio-Rad, Hercules, CA).
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