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 .
Af5198
Manufactured by Affinity Biosciences
AF5198 is a laboratory instrument designed for liquid handling applications. It is capable of automated pipetting and fluid transfer operations. The core function of AF5198 is to precisely dispense and aspirate liquids in a controlled and reproducible manner.
Automatically generated - may contain errors
Lab products found in correlation
Af0639, by Affinity Biosciences (2 mentions)
Af5393, by Affinity Biosciences (2 mentions)
Af7021, by Affinity Biosciences (1 mentions)
Af2002, by Affinity Biosciences (1 mentions)
Af7746, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Las4000 analyzer, by GE Healthcare (1 mentions)
Df7577, by Affinity Biosciences (1 mentions)
Af5103, by Affinity Biosciences (1 mentions)
Df6657, by Affinity Biosciences (1 mentions)
Af0199, by Affinity Biosciences (1 mentions)
2 protocols using af5198
1
Protein Expression Analysis in Cells
2
Western Blot Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Whole-protein extracts were obtained with sonication and separated on 10% SDS-PAGE and then were transferred onto PVDF membrane (Millipore, USA). Blocked with TBST (pH 7.5) containing 5% BSA, the membranes were incubated within primary antibodies against MCP1 (1 : 1200, DF7577, Affinity), iNOS (1 : 800, AF0199, Affinity), Arg1 (1 : 800, DF6657, Affinity), IL-1β (1 : 800, AF5103, Affinity), phosphorylated NF-κBp65 (1 : 1000, 3039, CST), NF-κBp65 (1 : 1000, 8242, CST), PPARγ (1 : 600, 2435, CST), HO-1 (1 : 1000, AF5393, Affinity), Nrf2 (1 : 1000, AF0639, Affinity), SOD1 (1 : 1000, AF5198, Affinity), the endogenous control β-actin (1 : 1000, 4970, CST), and GAPDH (1 : 1000, 3683, CST) at 4°C overnight. The membranes were then incubated in HRP-conjugated secondary antibodies (1 : 3000, 7074, CST) for 1 h at room temperature. We used the ECL Western Blot Kit (Thermo Scientific Pierce) to examine the bands and scanned them using a LAS4000 analyzer (GE Healthcare). The immunoblot density was examined by ImageJ and normalized by β-actin or GAPDH.
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