CRC cells were lysed on ice by lysis buffer (#KGP701, KeyGen Biotech, China) followed by quantificational measure with a bicinchoninic acid (BCA) Protein Assay Kit (#KGPBCA, KeyGen Biotech, China). Then, the lysates were subjected to SDS-polyacrylamide gel electrophoresis for separation. The isolated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (#IPVH00010, Millipore, USA). After fixing in methanol for 1 min, the PVDF membranes were blocked in PBST solution containing 5% nonfat milk and subsequently incubated overnight at 4°C with the following primary antibodies, respectively: anti-Akt (1:1,000 dilution, #YT0178, immunoway, USA), anti-p-Akt (Ser-473) (1:1,000, #YP0006, immunoway, USA), anti-Ezrin (1:5,000 dilution, #26056-1-AP, proteintech, USA), and anti-β-tubulin (1:5,000 dilution, #10094-1-AP, proteintech, USA). Then, the PVDF membranes were incubated with the secondary antibodies (Goat anti-rabbit IgG, 1:5,000 dilution, #BS13278, Bioworld, USA; Goat anti-mouse IgG, 1:5,000 dilution, #BS12478, Bioworld, USA) and were visualized with FDbio-Dura ECL Kit (#FD8020, Fdbio, China). All experiments were repeated three times and quantified using Gel-pro software.
Dura ecl kit
Manufactured by Fdbio
Sourced in China
The FDbio-Dura ECL Kit is a laboratory equipment product designed for chemiluminescent detection of western blots. The kit contains the necessary reagents for performing enhanced chemiluminescence (ECL) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg, by Bioworld Technology (1 mentions)
Goat anti mouse igg, by Bioworld Technology (1 mentions)
Lysis buffer, by Keygen Biotech (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Chemidoc touch, by Bio-Rad (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Sangon (1 mentions)
Chemidoc mp imager, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Ab32150, by Abcam (1 mentions)
Ab191217, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Bs13278, by Bioworld Technology (1 mentions)
Bs12478, by Bioworld Technology (1 mentions)
Yt3555, by Immunoway (1 mentions)
Yt2133, by Immunoway (1 mentions)
Ym3431, by Immunoway (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
7 protocols using dura ecl kit
1
Western Blot Analysis of Akt and Ezrin
2
Western Blot Protein Detection Protocol
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Tissues or cells were lysed in ice-cold cell lysis buffer (Cell Signaling Technology, Danvers, USA) containing 1x protease inhibitor (Beyotime, Shanghai, China). After centrifugation at 14,000 g for 10 min at 4 °C, total proteins were boiled in 1x SDS loading buffer at 100 °C for 10 min. Protein samples were then resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA; Sangon Biotech, Shanghai, China) in tris-buffered saline and Tween 20 (TBST) buffer for 1 h at the room temperature, the membranes were incubated with specific primary antibodies diluted in blocking buffer at 4 °C overnight. Next day, secondary antibodies conjugated to horseradish peroxidase (HRP) were used at room temperature for 1 h. Proteins were then detected with Fdbio-Dura ECL kit (Fdbio science, HangZhou, China) and quantified by Bio-Rad ChemiDoc Touch (Hercules, CA, USA).
3
Antibody Production and Detection Protocol
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The BmSUC1 antibody was produced as previously described (Daimon et al. 2008 (link)), and the PxSUC1 antibody was produced in the Huaan biotechnology company (Hangzhou, China) as previously described (Dai et al. 2019 (link)). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblot analyses were performed as previously described (Kiuchi et al. 2014 (link)). The anti-BmSUC1 or anti-PxSUC1 polyclonal antibody was used as the primary antibody (1:1,000), and the membrane was subsequently probed with goat antirabbit HRP-conjugated IgG (1:4,000) as the secondary antibody. Final signals were detected with FDbio-Dura ECL Kit (FD-bio science, China) using the chemiluminescence imaging system (Clinx, China).
4
Protein Expression Analysis by Western Blot
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Cells were harvested employing a western blotting and IP lysis buffer containing PMSF (Cat: P0013 and ST505, Beyotime Biotechnology, China). The protein concentration was quantified using The BCA protein assay (Thermo Fisher Scientific, USA). The protein samples were subsequently separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes. For the following steps, the membranes were blocked for an hour using a TBST solution containing 5% skim milk. Following this, the membranes were exposed to primary antibodies overnight at a temperature of 4 °C. Afterward, they were incubated with suitable HRP-conjugated secondary antibodies at a temperature of 25 °C for 60 min. Chemiluminescence was observed using a ChemiDoc-MP imager (Bio-Rad, Hercules, CA, USA) coupled with an FD Bio-Dura ECL Kit (Cat FD8020, Fdbio Science, China). The band density was analyzed using the ImageJ software. The primary antibodies used are listed in Supplement Table 2 . The suitable HRP-conjugated IgG secondary antibodies, namely Goat anti-rabbit (Cat: A0208) and anti-mouse (Cat: A0216), were procured from Beyotime Institute of Biotechnology, China.
5
Quantitative Protein Analysis by Western Blot
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The total protein was separated by SDS-PAGE electrophoresis and transferred onto PVDF membrane (#IPVH00010, Millipore, USA). Then, the PVDF membrane was incubated with the following primary antibodies at 4 °C overnight: anti-RRAGB (1:1000, 13,023–1-AP, Proteintech, USA), anti-mTOR (1:500, 66,888–1-IG, Proteintech, USA), anti-p-mTOR (Ser2448) (1:1000, YP0176, Immunoway, USA), anti-S6K (1:1000, YT3555, Immunoway, USA), anti-p-S6K (Thr389) (1:1000, YP1427, Immunoway, USA), anti-HIF1A (1:1000, YT2133, Immunoway, USA), anti-pro-caspase3 (1:1000, AB32150, Abcam, USA), anti-cleaved-caspase3 (1:500, YM3431, Immunoway, USA), anti-PARP (1:1000, AB191217, Abcam, USA), anti-cleaved-PARP (1:1000, AB32064, Abcam, USA), and anti-β-tubulin (1:2000, 10,094–1-AP, Proteintech, USA). Subsequently, the PVDF membrane was incubated with the corresponding HRP labeled goat anti-mouse (1:500, BS12478, Bioworld, USA) or goat anti-rabbit IgG (1:500, BS13278, Bioworld, USA) at room temperature for 1 h. FDBio-Dura ECL Kit (FD8020, FDbio, China) was used to visualize the protein bands.
6
Evaluating Protein Expression in Synovial Tissue
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Western blot analysis was performed to detect protein expression in synovial tissue samples and in in vitro experiments. Synovial tissue samples were frozen and powdered by grinding in liquid nitrogen and then thawed in radioimmunoprecipitation assay buffer supplemented with phenylmethylsulfonyl fluoride. A bicinchoninic acid protein assay kit (Beyotime) was used to quantify the proteins. Samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto PVDF membranes. Primary antibodies for the following proteins were used for immunodetection: GSDME (catalog no. ab215191; Abcam), activated caspase 3 (catalog no. 9661; Cell Signaling Technology), and β‐actin (catalog no. AP071; Bioworld Technology). After washing with Tris buffered saline–Tween, the probed membranes were incubated with HRP‐labeled secondary antibodies (HRP‐labeled goat anti‐rabbit IgG [heavy and light chains] [catalog no. 111‐035‐003; Jackson ImmunoResearch] and HRP‐labeled goat anti‐mouse IgG [heavy and light chains] [catalog no. 115‐035‐003; Jackson ImmunoResearch]) and then visualized with an FDbio‐Dura ECL Kit (FD‐bio science). Protein expression was analyzed using ImageJ software (National Institutes of Health).
7
Protein extraction and western blot analysis
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Cells were lysed and proteins were extracted on ice for 10 min with RIPA lysis buffer (EcoTop, China). Then, the proteins were boiled at 100 °C for 10 min with 5× loading buffer (FDbio, China). Proteins were separated by SDS-PAGE and were then transferred to 0.45 μm PVDF membranes. The membranes were blocked in 5% nonfat milk (for analysis of unphosphorylated proteins) or 5% BSA (for analysis phosphorylated proteins) before they were incubated with primary antibody. The membranes were incubated with secondary antibody the next day. The blots were visualized by an FDBio-Dura ECL Kit (FDbio, China). The antibodies are listed in Additional file 3: Table S3 . The data of western blot was quantitatively analyzed by ImageJ.
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