Total protein was extracted using RIPA Lysis Buffer (cat. no. AS1004; Aspen Biological) and Protease Inhibitor Cocktail (cat. no. 04693159001; Roche Diagnostics). The protein concentration was determined using the BCA Protein Quantification Kit (cat. no. AS1086; Aspen Biological). Subsequently, proteins (40 µg/lane) were separated using 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were then blocked with 5% skimmed milk and 0.1% Tween-20 in Tris-buffered saline at room temperature for 1 h. After which, the membranes were incubated overnight at 4°C with primary antibodies against APOA1 (1:1,000; cat. no. 14427-1-AP; Proteintech Group, Inc.) and β-actin (1:10,000; cat. no. TDY051; Beijing TDY Biotech Co., Ltd.). After washing three times with PBS containing 0.5% Tween-20, the membranes were incubated with HRP-conjugated anti-rabbit secondary antibodies (1:10,000; cat. no. AS1107; Aspen Biological) at room temperature for 1 h. Signal visualization was performed using a Lide110 scanner (Canon, Inc.).
Protease inhibitor cocktail
Manufactured by Aspen
Sourced in United States
Protease inhibitor cocktail is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It is commonly used in protein extraction and purification processes to prevent the degradation of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Aspen (2 mentions)
Ripa lysis reagent, by Aspen (2 mentions)
Bca protein assay reagent kit, by Aspen (2 mentions)
Sds loading buffer, by Aspen (2 mentions)
Ecl developer, by Aspen (2 mentions)
Camkii, by Proteintech (2 mentions)
Wnt5a, by Abcam (2 mentions)
Erk1 2, by Proteintech (2 mentions)
Stat3, by Proteintech (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Gapdh, by Proteintech (2 mentions)
Nf κb p65, by Proteintech (2 mentions)
α tubulin, by Proteintech (2 mentions)
Lide110 scanner, by Canon (1 mentions)
β actin, by Proteintech (1 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (1 mentions)
Ripa lysis buffer, by Aspen (1 mentions)
4 protocols using protease inhibitor cocktail
1
Quantification of APOA1 Protein by Western Blot
2
Smad7 Protein Extraction and Detection
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RIPA lysis buffer (ASPEN, NanTong, China) containing phenylmethylsulfonyl fluoride (Beyotime) and protease inhibitor cocktail (ASPEN, Pleasanton, CA, USA) were used to extract proteins. Then, the proteins were carried on a 10% SDS-PAGE gel via electrophoresis and transferred onto a polyvinylidene difluoride membrane. Five percent of skim milk was used to block the membrane for 2 hours and then together with an anti-Smad7 antibody were incubated (ab15116) for 24 hours and with secondary antibodies for 30 minutes. A developing agent was added to the membrane, and the results were recorded after X-ray exposure.
3
Western Blot Analysis of Signaling Pathways
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Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100 °C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4 °C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRP-conjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed five times for 30 min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 (1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII (1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).
4
Western Blot Protocol for Protein Analysis
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Total cellular proteins were extracted using RIPA lysis reagent (Aspen) containing a protease inhibitor cocktail (Aspen) with or without a phosphatase inhibitor (Bio-swamp). The protein concentration was measured by BCA protein assay reagent kit (Aspen) and mixed with 4 × SDS loading buffer (Bio-swamp) for denaturation in a 100°C boiling water bath for 8 min. The proteins were then separated on 10% SDS-PAGE gels and transferred to PVDF membranes (Merck). The PVDF membranes were blocked with 5% skim milk for 90 min, washed three times, and then incubated with primary antibody at 4°C overnight. Thereafter, the PVDF membranes were washed three times for 30 min and incubated with HRPconjugated secondary antibodies for 90 min at room temperature. Finally, the membranes were again washed ve times for 30min, exposed to ECL developer (Aspen) and analyzed by Bio-Rad Image Lab software. The following primary antibodies used in this study were listed: Wnt5a (1:1000, Abcam), ERK1/2 (1:1000, Protein Tech), p-ERK1/2 ( 1:1000, CST), STAT3 (1:1000, Protein Tech), p-STAT3 (1:1000, CST), NF-κB p65 (1:1000, Protein Tech), p-NF-κB p65 (1:1000, CST), CaMKII (1:1000, Protein Tech), p-CaMKII ( 1:1000, CST), GAPDH (1:5000, Protein Tech), α-Tubulin (1:5000, Protein Tech).
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