The total protein of Panc-1 and BxPC-3 cells was extracted by radioimmunoprecipitation assay buffer with protease inhibitor and the concentration of the protein was examined using the Bicinchoninic acid (BCA) Assay kit (Applygen Technologies Inc., Beijing, China). The protein of each sample was then separated by SDS-PAGE (Invitrogen Life Technologies, Carlsbad, CA, USA) and transferred onto the polyvinylidene difluoride membranes (Invitrogen Life Technologies). The membranes were blocked with 5% non-fat milk for 1 h and incubated with the primary antibodies at 4°C overnight. Next, the membranes were probed with the secondary antibodies for 1 h at room temperature. Immunopositive bands were then detected and exposed to film following incubation with an enhanced chemiluminescence system (Applygen Technologies, Inc., Beijing, China). The expression of protein was normalized to the levels of β-actin.
Enhanced chemiluminescence system
Manufactured by Applygen
Sourced in China
The Enhanced Chemiluminescence System is a laboratory equipment designed to detect and quantify proteins in Western blot analysis. It utilizes chemiluminescent substrates to produce light when they react with the targeted proteins, allowing for sensitive and accurate detection.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Bca assay kit, by Applygen (1 mentions)
Ab3578, by Abcam (1 mentions)
Image pro plus 6, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
6 protocols using enhanced chemiluminescence system
1
Western Blot Analysis of Panc-1 and BxPC-3 Cells
2
Immunoblotting Analysis of Ovarian Proteins
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Immunoblotting was performed as described previously (Wu et al. 2014) (link). RIPA lysis buffer was used to extract total protein from mice ovaries. In total, 40 μg of protein were separated in 10% Bis-Tris gels and then transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature (RT) with TBST (50 mM Tris-HCl, 150 mM NaCl and 0.1% (v/v) Tween-20) containing 5% (w/v) nonfat dried milk and then incubated with rabbit antimouse CYP11A1 (1:600, Proteintech Group, Chicago, IL, USA), rabbit antimouse StAR (1:300, Proteintech Group), rabbit anti-glucocorticoid receptor (1:1000, ab3578, Abcam) and mouse antimouse β-actin (1:2000, Zhongshan, Beijing, China ) antibodies overnight at 4°C. Membranes were then washed with TBST and incubated with HRP-labeled secondary antibody (1:10,000) for 1 h at room temperature. Protein bands on the membrane were visualized by an enhanced chemiluminescence system (Applygen Technologies Inc., Beijing, China) on X-ray film.
3
Western Blot Analysis of Liver Proteins
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Liver tissues were lysed in sample buffer containing 62 mM Tris-HCl, pH 6.8, 0.1% SDS, 0.1 mM sodium orthovanadate, and 50 mM sodium fluoride. The protein content was determined by the BCA protein assay. Equal amounts of proteins were loaded and separated by SDS-PAGE. After electrophoresis, the proteins were transferred on membranes, after being blocked with 3% nonfat dry milk the membrane with target proteins was incubated with an antibody against IRS-2, SREBP-1c, ACC, FAS, or GAPDH overnight at 4°C. The blots were incubated with a respective HRP-conjugated second antibody, and then immunoreactive bands were revealed using an enhanced chemiluminescence system (Applygen Technologies Inc., Beijing, China). The protein signal was quantified by scanning densitometry in the X-film by Image-Pro Plus 6.0 software (Bio-Rad, Hercules, California, USA) [24 (link)].
4
Western Blotting of Liver Proteins
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Total proteins in liver cells were extracted using RIPA lysis buffer (1% Triton X-100, 1% deoxycholate, 0.1% in sodium dodecyl sulfate) with 1 mM phenylmethanesulfonyl fluoride (PMSF). Nuclear protein was extracted from the liver tissues and protein concentration was measured by the BCA procedure. Then, 20µg protein samples were separated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto 0.45 µm (pore size) polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA). Membranes were blocked with 5% (m/v) skimmed milk at room temperature for 1 h. TLR4, CD14, and NF-κB antibodies diluted with 5% (m/v) bovine serum albumin at a ration of 1:200, IκBαa at 1:1000, β-Actin at 1:200 that was used to verify equal total protein, and then were placed at 4 °C for overnight. After washing with TBST three times, the membranes were incubated with secondary antibody goat anti-rabbit (at 1:5000) for 1 h at room temperature. The bands were detected using an enhanced chemiluminescence system (Applygen Technologies, Inc., Beijing, China) and were analyzed by Quantity One software.
5
Western Blot Analysis of COX-2 Expression
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Equal amounts of lysates were loaded onto a 12% SDS-PAGE following a heat-induced denaturation for 5 min at 100°C. The separated protein was then transferred into PVDF membranes. The membranes were then blocked with 5% BSA in TBS-T and incubated with primary antibodies against COX-2 or β-actin at 4°C overnight. The membranes were washed with TBS-T three times and incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Finally, signals were detected using an enhanced chemiluminescence system (Applygen Technologies, Beijing, China), and Image J 1.47 software was used to calculate integrated optical density of protein bands.
6
Western Blot Analysis of Protein Targets
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Total protein was resolved by SDS-polyacrylamide gel electrophoresis [30 (link)] and then was transferred to nitrocellulose membranes. After blocking in 5% bovine serum albumin (BSA) at 25°C for 1 h, the membranes were washed 3 times with TBST for 15 min each time and incubated at 4°C overnight with primary antibodies against MPO (1 : 500), DAG (1 : 500), RAF (1 : 500), gpr84 (1 : 500), PKC (1 : 2000), MAPK3 (1 : 2000), caspase 8 (1 : 500), RIP1 (1 : 500), RIP3 (1 : 500), MLKL (1 : 500), and GAPDH (1 : 5000, Santa Cruz Biotechnologies, CA, USA). Then, the membranes were washed in TBST 3 times, incubated with a secondary antibody at 25°C for 1 h, and washed in TBST 3 times. Finally, the protein band signals were examined using an enhanced chemiluminescence system (Applygen Technologies Inc., Beijing, China) and analyzed by ImageJ software (National Institutes of Health). GADPH was used as a loading control.
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