The ATDC5 cells at the concentration of approximately 3 × 105 were seeded in 6-well plates. The cell density was reached 80% before transfected with plasmids. 48 h after transfection, cells cultured medium was removed and RIPA was added to extract protein. The concentration of protein was measured by BCA protein quantification assay kit (Beyotime, Shanghai). Then proteins samples were separated by SDS-PAGE (5% of concentration gel and 12% of separation gel) and then transferred to PVDF membranes. The membranes were blocked by 5% skimmed milk for 1.5 h and incubated with primary antibodies [DDX3X, P65, p-P65, IkappaB (IkB-α), p-IkappaB (p-IkB-α), MMP-3, MMP-13]at 1:1000 dilution overnight at 4 °C. After washing the bands for three times with TBST, the secondary antibody of goat anti-rabbit was used to incubated for 1 h at 1:10,000 dilution at room temperature. According to the manufacturer’s protocol, the protein membranes were detected with ECL immunoblotting kit (Millipore, USA).
Ecl immunoblotting kit
Manufactured by Merck Group
Sourced in United States
The ECL immunoblotting kit is a laboratory equipment product that enables the detection and quantification of specific proteins in a sample using a chemiluminescent detection method.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (9 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (4 mentions)
Bca detection kit, by Keygen Biotech (3 mentions)
Bca protein quantification assay kit, by Beyotime (1 mentions)
Pvdf membrane, by Abcam (1 mentions)
Peg10, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti igf1r antibody, by Abcam (1 mentions)
Stard13, by Proteintech (1 mentions)
Ripa buffer, by Transgene (1 mentions)
Ab126705, by Abcam (1 mentions)
11 protocols using ecl immunoblotting kit
1
Western Blot Analysis of ATDC5 Cells
2
Protein Extraction and Western Blot Analysis
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The total protein from the cultured HCC cells and the tissue samples was isolated by RIPA (Beyotime, Haimen China). Proteases and phosphatases were added to RIPA to prevent protein degradation. BCA detection Kit (Keygen, Nanjing, China) was used for qualification according to the manufacturer’s protocol. Protein samples were loaded for electrophoresis (5% gel for concentration and 10% for separation). Following electrophoresis, the proteins were transferred on a PVDF membrane (Merck Millipore) and were blocked by 5% nonfat milk for 1 h. Then the PVDF membrane was incubated overnight at 4 °C with the primary antibodies (PEG10; Abcam) at 1:1000 dilution. The next day, the membrane was incubated with secondary antibodies (a dilution rate of 1:5000; Zhuangzhi Biology, China) for 1 h at room temperature. The protein bands were evaluated by ECL immunoblotting kit following the manufacturer’s protocol (Millipore, USA).
3
Protein Expression Analysis in HCC
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After transfection, total proteins were extracted from HCC tissues and cells using RIPA lysis buffer (Beyotime, Beijing, China). Protein concentrations were calculated by BCA Protein Assay Kit (Beyotime, Beijing, China). Then, equal amount of protein samples were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane (Invitrogen). Next, membrane was blocking with 5% non-fat milk for 1 hour at room temperature and incubated with primary antibodies (anti-IGF1R antibody, 1:1000, Abcam; anti-GAPDH antibody, 1:10 000 Abcam) overnight at 4°C. In addition, the membrane was incubated with corresponding secondary antibodies at room temperature for 1 hour. The immunoreactive signal was detected by an ECL immunoblotting kit (Millipore, USA).
4
Western Blot Analysis of Protein Expression
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The transfected cells were lysed by using RIPA (Beyotime) supplemented with proteinase and phosphatase inhibitors. Protein samples were loaded for electrophoresis (5% gel for concentration and 10% for separation), and then transferred onto 0.45 μm or 0.22 μm pore size PVDF membrane (Merck Millipore). After blocking with 5% non‐fat milk for 1 hour, the membrane was incubated with specific primary antibodies (Supplementary Table 2) at 4°C overnight. The next day washed the membrane and then incubated with secondary antibodies (Zhuangzhi Biology, dilution rate of 1:5000) for 1 hour at room temperature. Proteins bands were detected by using ECL immunoblotting kit (Millipore) according to the manufacturer's protocol.
5
Western Blotting Analysis of Proteins
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All proteins were isolated after transfection for 72 hours as the protocol of radioimmunoprecipitation assay (RIPA) lysis buffer (Pierce) which was added proteinase and phosphatase inhibitors in advance. Protein samples were loaded for electrophoresis (5% gel for concentration and 10% for separation) and then transferred onto 0.45 μm or 0.22 μm pore size PVDF membrane (Merck Millipore). After blocking with 5% defatted milk gently for 1 hour, the membrane was incubated with corresponding primary antibodies (Table 3 ) at 4°C overnight. The next day washed the membrane and then incubated with secondary antibodies (Zhuangzhi Biology, dilution rate of 1:5000) for 1 hour at room temperature. Proteins bands were detected by using ECL immunoblotting kit (Millipore). For convenience, the raw data of Western blot were supplied in Figures S1‐S3 for bands involved in Figures 5 J, 7 F and 8 in sequence.
6
Western Blot Protein Analysis
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After transfection for 48 h, proteins were collected from cells using RIPA Buffer (Pierce) containing proteinase and phosphatase inhibitors. Each protein sample was electrophoresed with SDS-polyacrylamide gel electrophoresis (4% stacking and 10% SDS–PAGE separating gels), transferred to the PVDF membrane (Merck Millipore). The membrane was blocked with 5% non-fat milk and incubated with corresponding primary antibodies (Supplementary Table 2 ) at 4 °C overnight. Then washed the membrane extensively and followed by incubation with secondary antibodies (Zhuangzhi Biology, China) at room temperature for 1 h. The results were subsequently subjected to immunoblotting analysis using the ECL immunoblotting kit (Millipore, USA) according to the manufacturer’s protocol. Image J software-based analysis was used to quantify the bands obtained through Western blot (Supplementary figures ).
7
STARD13 Protein Expression Analysis
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After transfection for 72 h, all proteins were isolated by RIPA (Pierce). Proteases and phosphatases were added to RIPA in advance to prevent protein degradation. Protein samples were loaded for electrophoresis (5% gel for concentration and 10% for separation), and then the proteins were transferred on a 0.45 μm or 0.22 μm pore size PVDF membrane (Merck Millipore). After blocking with 5% defatted milk gently for 1 h, the membrane was incubated at 4 °C overnight with primary antibodies (STARD13; Proteintech) at 1:2000 dilution. The next day, the membrane was washed extensively and incubated with secondary antibodies (at 1:2500 dilution; Zhuangzhi Biology, China) for 1 h at room temperature. Proteins bands were detected by using ECL immunoblotting kit (Millipore, USA) following the manufacturer’s protocol.
8
Western Blot Analysis of Cultured HCC Cells
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The total protein from the cultured HCC cells and the tissue samples was isolated by RIPA (Beyotime, Haimen, China) supplemented with proteinase and phosphatase inhibitors. BCA detection kit (Keygen, Nanjing, China) was used for the quantification according to the manufacturer’s protocol. During electrophoresis, 5% gel for concentration and 10% for separation were used. The proteins were then transferred on a PVDF membrane (Merck Millipore) and were blocked by 5% nonfat milk for 1 h. Then, the PVDF membrane was incubated overnight at 40C with the primary antibodies (Supplementary Table 2 ). On the next day the secondary antibody (Zhuangzhi Biology, China) was diluted in TBST in a 1:5000 ratio, and the membranes were re-incubated for 1 h. The protein bands were evaluated by ECL immunoblotting kit following the manufacturer’s protocol (Millipore, USA).
9
Protein Expression Analysis in Colon Cells
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Proteins were extracted from treated NCM460 and DLD-1 cells using RIPA Buffer (TransGen, Beijing, China) after transfection for 36 h with proteinase. The protein samples were separated by SDS-PAGE (polyacrylamide gel electrophoresis), and then transferred to PVDF membranes (Merck Millipore). Next, 5% non-fat milk was used to block the transferred PVDF membrane and incubated with TGR5 (ab72608), Akt (ab8805), p65 (ab16502), and Bcl-2 (ab32124) separately at 4°C overnight. All the transferred membranes were washed extensively and then were incubated with corresponding secondary antibodies (TransGen, Beijing, China) for 1 h at room temperature on the next day. The results are from immunoblotting detection using the ECL immunoblotting kit (Millipore, USA).
10
Protein Isolation and Western Blot Analysis
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The total protein from the cultured HCC cells and the tissue samples were isolated by RIPA (Beyotime, Haimen China) supplemented with proteinase and phosphatase. BCA detection kit (Keygen, Nanjing, China) was used for quali cation according to the manufacture's protocol. For electrophoresis 5% gel was used for concentration and 10% for separation. Following electrophoresis the proteins were transferred on a PVDF membrane (Merck Milipore) and were blocked by 5% non-fat milk for 1 hour. Then the PVDF membrane was incubated overnight at 4 0 C with the primary antibodies (Supplementary Table 2). On the next day the secondary antibody (Zhuangzhi Biology, China) was diluted 1:5000 ratio in TBST, and the membrane was re-incubated for 1 hour. The protein bands were evaluated by ECL immunoblotting kit following the manufacture's protocol ( Milipore, USA).
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