FU97 cell lysates were extracted using RIPA Lysis Buffer containing protease and phosphatase inhibitors (Beyotime), fractionated by SDS‐polyacrylamide gel electrophoresis, and electrotransferred to polyvinylidene difluoride (PVDF) membranes. After blocking in 5% skim milk in triethanolamine buffered saline with 1‰ Tween‐20 (TBST) for 1 h at room temperature, the membranes were incubated with primary antibodies (AFP [1:2500, Abcam, EPR20667], GPC3 [1:1000, HUABIO, EM1709‐60], ALB [1:500, HUABIO, ET1702‐55], E‐cadherin [1:10000, Abcam, EP700Y], N‐cadherin [1:5000, Abcam, EPR1791‐4], VIM [1:1000, Abcam, EPR3776], SNAIL [1:1000, Abcam, EPR21043], and GAPDH [1:5000, Abcam, EPR16891]) at 4°C overnight and then with peroxidase‐conjugated secondary antibodies (HRP Conjugated Goat Anti‐Rabbit IgG [1:5000, HUABIO]) for 1 h at room temperature. After washing thrice with TBST, the blot was soaked in Super ECL Detection Reagent (YEASEN) for 10 s. The membranes were then exposed to a film (Kodak) for 10 s in the dark.
Ripa lysis buffer containing protease and phosphatase inhibitors
Manufactured by Beyotime
Sourced in China
RIPA lysis buffer containing protease and phosphatase inhibitors is a solution used for cell lysis and protein extraction. It aids in the solubilization of cellular proteins while preserving their post-translational modifications. The buffer includes a blend of detergents, salts, and inhibitors to prevent protein degradation by proteases and dephosphorylation by phosphatases.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Beyotime (3 mentions)
Hrp conjugated secondary antibody, by Absin (3 mentions)
Multifunctional microplate reader, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Epr3776, by Abcam (1 mentions)
Ep700y, by Abcam (1 mentions)
Epr16891, by Abcam (1 mentions)
Epr1791 4, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Huabio (1 mentions)
Super ecl detection reagent, by Yeasen (1 mentions)
4 protocols using ripa lysis buffer containing protease and phosphatase inhibitors
1
Western Blot Analysis of Liver Cancer Markers
2
Evaluating Anti-Proliferative Effects of SAHA Formulations
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The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x10 4 cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China).
2.9 Western blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).
2.9 Western blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).
3
Anti-proliferative Effects of SAHA and Nanomicelles
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The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x10 4 cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China). 2.10 Protein blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).
4
Anti-proliferative Effects of SAHA Formulations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The anti-proliferative effects of SAHA, SAHA-loaded nanomicelles and empty nanomicelles were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay (Aladdin, China). 1x10 4 cells/well were seeded in 96 well plates, grown overnight, and then treated with various concentrations of SAHA, SAHA loaded nanomicelles and empty nanomicelles for 24 h, 48 h or 72 h. 20 μL of MTT reagent were added to each well and left incubating for 4 hours. The optical density was determined at 490 nm using a Multifunctional Microplate Reader (Thermo Fisher, China).
2.10 Protein blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).
2.10 Protein blot 2.5x10 5 cells were dispersed in three 6-well plates, grown overnight, and three plates treated with SAHA, SAHA nanomicelles and empty nanomicelles for 24 h or 48 h. The cells were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime, China) and total protein was estimated with BCA Protein Assay Kit (Beyotime, China). Protein was separated by SDS-PAGE and transferred on PVDF membranes (Beyotime, China). The membranes were blocked in 5% skimmed milk, incubated with primary antibodies for p21, p53, N-Cadherin or E-cadherin (Santac Cruz, US), and then incubated with the appropriate HRP conjugated secondary antibody (Absin, China).
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