Frozen colon tissue was homogenized in RIPA buffer, samples were centrifuged, and the supernatant was collected. The quantification of total protein was performed using a bicinchoninic acid assay. After denaturation and adjustment of the concentration, the protein samples were separated by electrophoresis and transferred onto protein membranes. Subsequently, the membranes were blocked in 5% skim milk solution for 1 h at room temperature, followed by overnight incubation with primary antibodies (anti-5-HT4R, 1 : 1000, ab60359, Abcam; anti-PKA, 1 : 1000, AF7746, Affinity; anti-SP, 1 : 2000, DF7522, Affinity; or anti-CGRP, 1 : 1000, ab189786, Abcam) on a shaker at 4°C. After being washed, the membranes were further incubated with secondary antibodies at room temperature for 1 h. Finally, the membranes were washed, and protein bands were visualized and imaged using a Bio-Rad imaging system. The gray values of individual protein bands were quantified using ImageJ software.
Af7746
Manufactured by Affinity Biosciences
Sourced in China
The AF7746 is a sensitive and versatile lab instrument for detecting and quantifying specific biomolecules in samples. It utilizes advanced detection techniques to provide accurate and reliable results. The core function of the AF7746 is to facilitate the analysis of various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab189786, by Abcam (2 mentions)
Ab60359, by Abcam (2 mentions)
Df7522, by Affinity Biosciences (2 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Af5198, by Affinity Biosciences (1 mentions)
Af2002, by Affinity Biosciences (1 mentions)
Af0639, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Af5393, by Affinity Biosciences (1 mentions)
Tb green premix ex taq tli rnaseh plus, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser perfect, by Takara Bio (1 mentions)
Sabc hrp kit, by Beyotime (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Df2267, by Affinity Biosciences (1 mentions)
Bca kit, by Keygen Biotech (1 mentions)
Af7011, by Affinity Biosciences (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
4 protocols using af7746
1
Protein Expression Analysis in Colon Tissue
2
Protein Expression Analysis in Cells
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After treatment, the cells were harvested and lysed using a cell buffer (WB038, GEFAN, Nanjing, China), and their concentrations were estimated by a BCA kit (GM03, GEFAN). After denaturation, cells were subjected to protein electrophoresis. After transferring to a PVDF membrane (WB041, GEFAN), the membrane was sealed with 5% skim milk. After 2 h, cells were reacted with primary antibodies overnight at 4°C followed by incubation with a goat anti-rabbit secondary antibody IgG H&L (1:3000, S0001, Affinity Biosciences, Cincinnati, OH, USA) for 1 h at 37°C. Finally, a color reagent (E266188, Aladdin, Shanghai, China) was used to visualize the bound antibodies, which were then placed in a chemiluminescent instrument (610020-9Q, Clinx, Shanghai, China). The primary antibodies of the rabbit anti-human Nrf2 (1:2000, AF0639), SOD-1 (1:1500, AF5198), CAT (1:2000, AF7746), HO-1 (1:1500, AF5393), nuclear factor kappa B (NF- κB, 1:1500, AF5006), phospho-IkappaB-alpha (p-IKBα, 1:2000, AF2002), and GAPDH (1:20000, AF7021) were obtained from Affinity Biosciences .
3
Mosapride Citrate Modulates 5-HT4R Pathway
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Activated carbon powder and gum arabic were obtained from Shanghai Sangon Biological Engineering Technology and Service; mosapride citrate dispersible tablets were obtained from Chengdu Kanghong Pharmaceutical Group (Sichuan Province, China); the PrimeScript™ RT Reagent kit with gDNA Eraser (Perfect Real Time, RR047A) and TB Green® Premix Ex Taq™ (Tli RNase H Plus) were obtained from Takara; the SABC-HRP Kit (P0612) was obtained from Beyotime Biotechnology (Jiangsu Province, China); anti-5-HT4R antibody (1 : 200 for immunohistochemistry (IHC) and 1 : 1000 for western blot (WB), ab60359) was obtained from Abcam; anti-PKA antibody (1 : 1000, AF7746) and anti-SP antibody (1 : 2000, DF7522) were obtained from Affinity; anti-CGRP antibody (1 : 1000, ab189786) was obtained from Abcam; and the 5-HT enzyme-linked immunosorbent assay (ELISA) kit (SDM0131) and the cAMP ELISA kit (SDM0130) were obtained from Simuwu Biotechnology (Shanghai, China).
4
Colon Tissue Protein Analysis
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Total protein was extracted from the proximal colon tissues with RIPA buffer containing PMSF and determined by a BCA kit (Keygen Biotech, KGP902, China). The protein samples (60 μg/lane) were loaded on SDS-PAGE gels (Beyotim, P0012A, China) and transferred onto PVDF membranes (Merck, IPVH00010, United States) after electrophoresis. Membranes were blocked with 5% skimmed milk for 2 h and then incubated overnight at 4°C with CACNA1C (1:500, Affinity, DF2267), CACNA1D (1:500, Affinity, DF2267), cyclic adenosine monophosphate (cAMP, 1:10000, Abcam, ab76238), protein kinase A (PKA, 1:500, Affinity, AF7746), and β-Tubulin (1:5000, Affinity, AF7011) antibodies. Afterward, an HRP-conjugated antibody (1:5000, Affinity, S0001, China) were incubated with the membrane. The chemiluminescence (ECL) kit was employed for the membrane visualization by a Tanon 5200 system (Shanghai, China). ImageJ 7.0 was used for quantification.
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