Equal amounts of total prostatic protein (30 µg) were heated at 100 °C for 5 min, loaded onto 10–12% SDS-PAGE, and electrophoresed. The proteins were then transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore Corporation, Burlington, MA, USA) using a Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). After blocking nonspecific binding sites with 5% nonfat milk at room temperature for 1 h, the membranes were incubated with the primary antibodies overnight at 4 °C. The immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA) with exposure to an image analysis system (AI680, GE Healthcare, Uppsala, Sweden).
Ai680
Manufactured by GE Healthcare
Sourced in Sweden
The AI680 is a high-performance laboratory equipment designed for advanced scientific analysis. It utilizes cutting-edge artificial intelligence technology to enhance data processing and analysis capabilities. The core function of the AI680 is to provide researchers and scientists with a powerful tool for conducting comprehensive and efficient experiments.
Automatically generated - may contain errors
Lab products found in correlation
Transfer system, by Bio-Rad (2 mentions)
Immobilon p polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
Polyvinylidene difluoride filter membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Bolt mes, by Thermo Fisher Scientific (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Light sensitive film, by Cytiva (1 mentions)
Anti p62, by Cell Signaling Technology (1 mentions)
Anti e cadherin, by ABclonal (1 mentions)
Nitrocellulose nc membrane, by GE Healthcare (1 mentions)
Anti vimentin, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Tomm20, by Abcam (1 mentions)
Alpha tubulin, by Merck Group (1 mentions)
Cleaved caspase 7, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
7 protocols using ai680
1
Western Blot Protein Analysis
2
SDS-PAGE and Western Blotting Protocol
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SDS–PAGE was performed with 8% or 10% acrylamide gels and BOLT MES (Invitrogen) or Tris-glycine SDS running buffer. Western blotting was performed using polyvinylidene difluoride filter membranes (Millipore) for electro-transfer and Blocking One (Nacalai Tesque) for blocking. Signals were developed with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Bio-Sciences) or peroxidase-conjugated primary antibodies using Western Lightning Plus-ECL (PerkinElmer) and the chemiluminescent system AI680 (GE Healthcare) as described (Miyata & Nishida, 2004 (link)). In some experiments, polyvinylidene difluoride membrane wetting was performed with ethanol instead of methanol, and we confirmed that this does not affect the results. For all the Western blotting data, we repeated independent experiments and showed representative images of obtained consistent results.
3
Western Blot Protocol for Protein Analysis
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Equal amounts of total prostatic protein (30 µg) were heated at 100 °C for 5 min, loaded onto 10–12% SDS-PAGE and electrophoresed. The proteins were then transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore Corporation, Burlington, MA, USA) using Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). After blocking nonspecific binding sites with 5% nonfat milk at room temperature for 1 h, membranes were incubated with the primary antibodies overnight at 4 °C. The immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies, using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA) with exposure to light-sensitive film (Amersham Pharmacia, Piscataway, NJ, USA). The band intensity was determined by a computer image analysis system (AI680, GE Healthcare, Uppsala, Sweden).
4
Protein Expression Analysis in Cell Samples
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Cells were scraped and homogenized with Sample Buffer, Laemmli 2x Concentrate (S3401; SIGMA). Protein per sample was separated by polyacrylamide gel electrophoresis and then transferred to nitrocellulose (NC) membrane (GE Healthcare, Piscataway, NJ, USA) and detected with the antibodies. The signals were scanned by AI 680 (GE). Anti-p53 (no.2527S), Anti-Snail (no.3895S), Anti-N-cadherin (no.13116S), Anti-Ubiquitin (no.3933S), Anti-LC3 A/B (no.12741S) and Anti-p62 (no.88588S) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-mdm2 (no. A0345) and Anti-E-cadherin (no.A3044) were purchased from Abclonal (Wuhan, China). Anti-β-Actin (no. A1978) and anti-Vimentin (no.V6630) were purchased from Sigma (Sigma, Victoria, BC, Canada).
5
Protein Expression and Detection in Cell Lysates
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Cells were collected and lysed in RIPA buffer (Biosolution, BR002) containing HaltTM Protease Inhibitor Cocktail (Roche, 34044100). Proteins were separated using 7-15% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (GE Healthcare, 10600002). The following primary antibodies were used: CRBN (Atlas antibodies, HPA045910), Flag (Sigma-Aldrich, F3165), MFN2 (Abcam, ab50843), cytochrome C (Santa Cruz, sc13156), TOMM20 (Abcam, ab56783), alpha-tubulin (Sigma-Aldrich, T5168), cleaved caspase-7 (Cell signaling, #9491), cleaved PARP (Cell signaling, #9541L), p53 (Santa Cruz, sc126), p21 (Cell signaling, 2947S) and beta-actin (Santa Cruz, sc47778). Membranes were visualized using Western Pico or Femto ECL solution (ThermoFisher Scientific, 34580, 34096) on a chemiluminescent image analyzer (GE Healthcare, AI680).
6
Protein Analysis of HUVEC and Tumor Tissue
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HUVECs were washed twice with ice-cold PBS and lysed with cell lysis buffer (Beyotime Biotechnology) containing an EDTA-free protease inhibitor (Roche). Tumor tissue was incubated with ice-cold cell lysis buffer containing protease inhibitor and disrupted with Tissuelyser-24. Protein concentration was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed as previously described (23 (link)). Blots were incubated with Pierce ™ ECL Western Blotting Substrate (Thermo Fisher), and detected by AI 680 (General Electric Company, Boston, MA) Antibodies used for western blotting included rabbit anti-phospho-STAT6 (Tyr-641, 1:1000, Cell Signaling Technology); rabbit anti-STAT6 (1:1000, Abcam); rabbit anti-NRP1 (1:1000, Affinity); rabbit anti-NRP1 (1:1000, Abcam); and rabbit anti-Tubulin (1:5000, Proteintech). The secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:8000, Proteintech).
7
Western Blot Analysis of Protein Samples
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Protein samples were extracted as explained in our previous work [20] (link). Then, the protein concentration of the cell lysate was measured using a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equivalent amounts of denatured cell lysate (30 µg of cell lysate per sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The membrane was incubated with the primary antibody diluted in 5% skim milk powder in PBS containing 0.05% Tween 20 (PBST) overnight at 4 • C.
Following primary antibody incubation, the membranes were washed and incubated in the HRP-conjugated secondary antibody diluted in 5% skim milk in PBST for 1 h at room temperature. Protein expression was analyzed using an enhanced chemiluminescence (ECL) detection system (AI680, GE Healthcare, Uppsala, Sweden).
Following primary antibody incubation, the membranes were washed and incubated in the HRP-conjugated secondary antibody diluted in 5% skim milk in PBST for 1 h at room temperature. Protein expression was analyzed using an enhanced chemiluminescence (ECL) detection system (AI680, GE Healthcare, Uppsala, Sweden).
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