RIPA buffer containing PMSF (Beijing Solarbio Science & Technology Co., Ltd.) was used to extract total protein from transfected cells. The quantification of protein was determined using the BCA Protein Assay Kit [Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.]. The lysates (40 µg) were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (MilliporeSigma). Then, membranes were blocked with 5% skimmed milk at room temperature for 60 min. Following which, the membranes were incubated overnight at 4°C with specific primary antibodies against: β-actin (1:10,000; cat. no. AC026; ABclonal Biotech Co., Ltd.), E-cadherin (1:1,000; cat. no. E-AB-31261; Elabscience Biotechnology, Inc.), N-cadherin (1:1,000; cat. no. E-AB-64011; Elabscience Biotechnology, Inc.), Vimentin (1:1,000; cat. no. bs-0756R; BIOSS), Twist (1:1,000; cat. no. bs-2441R; BIOSS). After washing, the membranes were incubated at room temperature for 45 min with the appropriate IgG HRP-conjugated secondary antibody (1:10,000; cat. no. SA00001-9; ProteinTech Group, Inc.) and detected with enhanced chemiluminescence (ECL) reagent [Hangzhou Multi Sciences (Lianke) Biotech Co., Ltd.] and analyzed using ImageJ software (version 1.48; National Institutes of Health).
Twist
Manufactured by Bioss Antibodies
The Twist is a laboratory instrument designed for nucleic acid manipulation and analysis. It facilitates the preparation and processing of DNA and RNA samples for various applications, such as sequencing and molecular biology experiments. The core function of the Twist is to automate and streamline common laboratory workflows, enabling efficient and consistent sample handling.
Automatically generated - may contain errors
Lab products found in correlation
N cadherin, by Elabscience (1 mentions)
Vimentin, by Elabscience (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
E cadherin, by ABclonal (1 mentions)
N cadherin, by Bioss Antibodies (1 mentions)
Snail, by Bioss Antibodies (1 mentions)
Pierce ecl western blotting substrate, by GE Healthcare (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Beyotime (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
2 protocols using twist
1
Western Blot Analysis of EMT Markers
2
Protein Expression Analysis via Western Blot
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The proteins were isolated from cells, separated by 10% SDS–PAGE, and transferred onto PVDF membranes. Then, the membranes were blocked with 5% BSA for 2 h and incubated with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature, respectively. Anti-ABCC1 (1:1,000, bs-24241R), Anti-ABCB1 (1:1,000, bs-0563R), Anti-ERCC1 (1:1,000, bs-1726R), Snail (1:2,000, bs-1371R), and Twist (1:2,000, bs-2441R) were obtained from Bioss (Bioss, BeiJing); N-cadherin (1:1,000, 14215S), E-cadherin (1:1,000, 14472S), Slug (1:2,000, 9585T), Stat3 (1:1,000, 9139T), and p-Stat3 (1:1,000, 4113S) were obtained from Cell Signaling Technology (MA); β-actin (1:5,000, 81115-1-RR) was obtained from Proteintech (MA); Goat-anti-mouse IgG (1:5,000, A0216) was obtained from beyotime (Shanghai). Membranes were exposed to Pierce ECL Western Blotting Substrate (GE Healthcare). Band intensities were determined using ImageJ (National Institutes of Health). The band intensities were represented by the averages of three independent experiments.
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