Fluorescein isothiocyanate (FITC)-conjugated elastin (AnaSpec; 10 μg/ml) was incubated with 7 nM cdMMP-12 D4/wt and 2–2,000 nM purified Fab in 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl2, 100 μM ZnCl2 pH 7.5. Generated fluorescent signals were measured at 12 hr with λEx of 490 nm and λEm of 520 nm. For inhibition assays on fibronectin, 200 nM fibronectin (Corning) was incubated with 200–350 nM cdMMP-12 wt and 2 μM GM6001 or 0.1–6 μM Fabs in 50 mM HEPES, 150 mM NaCl pH 7.5 at 37°C for 2–4 hr. The samples were separated on 5% nonreducing SDS-PAGE and analyzed by western blot analysis with anti-fibronectin antibody (Abcam) and chemiluminescent substrate (Thermo Fisher Scientific). For inhibition assays on modified TEM-1 carrying cleavable sequence (PLGLEEAK) and an N-terminal HA tag, 250-nM modified TEM-1 was incubated with 700 nM cdMMP-12 wt and 10 μM Fabs in 50 mM HEPES, 150 mM NaCl pH 7.5 at 37°C for 4 hr. The samples were separated on 12% nonreducing SDS-PAGE, and the modified TEM-1 was visualized by western blot analysis using anti-HA antibody (Invitrogen) and chemiluminescent substrate. Assays without cdMMP-12s or Fabs were performed as controls.
Chemiluminescent substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, United Kingdom
Chemiluminescent substrate is a reagent used in molecular biology and biochemistry applications to detect and quantify specific biomolecules, such as proteins or nucleic acids. It generates a luminescent signal upon interaction with a target analyte, enabling sensitive and quantitative detection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (24 mentions)
Nitrocellulose membrane, by Bio-Rad (19 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (19 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Protease inhibitor cocktail, by Roche (14 mentions)
β actin, by Cell Signaling Technology (13 mentions)
Ripa buffer, by Merck Group (13 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (13 mentions)
Ripa buffer, by Thermo Fisher Scientific (12 mentions)
Chemidoc imaging system, by Bio-Rad (10 mentions)
Bca kit, by Thermo Fisher Scientific (10 mentions)
Pvdf membrane, by Bio-Rad (8 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (8 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (7 mentions)
Chemidoc mp imaging system, by Bio-Rad (7 mentions)
Nitrocellulose membrane, by Merck Group (7 mentions)
Alkaline phosphatase conjugated secondary antibody, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Abcam (6 mentions)
Anti gapdh, by Cell Signaling Technology (6 mentions)
Ripa buffer, by Beyotime (6 mentions)
358 protocols using chemiluminescent substrate
1
Inhibition of MMP-12 by Fab Fragments
2
Western Blot Protocol for Protein Analysis
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PBS washed cells were lysed in CelLytic buffer (Sigma-Aldrich) supplemented with 1% protease inhibitor cocktail (Roche Diagnostics), 1% phosphatase inhibitor cocktail (EMD Millipore) and 1mM PMSF on ice. A total 10 μg of cell lysates were separated on mini precast gels (Bio-Rad) and transferred on nitrocellulose membranes (Bio-Rad). Membranes were probed with the appropriate primary antibodies and were incubated with alkaline-phosphatase conjugated secondary antibodies (Invitrogen) and chemiluminescent substrate (Invitrogen) and were further detected by film exposure, UVP BioSpectrum 810 Imaging System (Thermo Fisher Scientific) or by using the ChemiDoc MP Imaging System (BioRad). Antibodies used in this study in Supplementary Table S10 with 1:750 to 1:1000 dilution in blocking buffer.
3
Western Blot Analysis of DNMT3A
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Whole cells were lysed in the RIPA lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics, USA). Samples were separated using SDS-PAGE (Bio-Rad, USA) and transferred onto nitrocellulose membranes (Bio-Rad, USA). Primary antibodies, including anti-DNMT3A (3598S, Cell Signaling, USA, 1:1000), anti-β-actin (A2066, Sigma-Aldrich, Sweden, 1:1000), as well as horseradish peroxidase-conjugated secondary antibodies (7074, Cell Signaling, USA, 1:1000), and chemiluminescent substrate (Invitrogen, USA) were used.
4
Protein Expression Analysis by Western Blot
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Total cell lysates were homogenized in tissue lysis buffer (FNN0071; Invitrogen) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland).20 (link) Proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Membranes were then incubated with primary antibodies against alpha-smooth muscle actin (αSMA) (diluted 1:500; Abcam, Cambridge, UK), tissue inhibitor of metalloproteinase (TIMP)1 (diluted 1:500; Invitrogen), and β-actin (diluted 1:2,000; Peprotech, Rocky Hill, NJ, USA) overnight at 4°C. On the following day, the membranes were incubated with the appropriate secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) and proteins were visualized using a chemiluminescent substrate (Invitrogen).
5
Fractionation and Western Blot Analysis
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Cell lysates were prepared with RIPA buffer (Sigma-Aldrich) using the standard protocol. Nuclear and cytoplasmic fraction analysis from the THP-1 cells was performed with nuclear and cytoplasmic extraction reagents (Thermo Scientific) according to the manufacturer’s protocol. Briefly, the harvested cells were suspended in lysis buffer containing a proteinase inhibitor cocktail (Sigma-Aldrich) and centrifuged at 16,000 × g for 5 m. The supernatant was saved for cytoplasmic extracts, and the pellet was re-suspended in a special buffer (NER) for the nuclear fraction. After being centrifuged at 16,000 × g for 10 m, the supernatant was collected for nuclear extract. The collected supernatant from the total cell lysates or the cytosol and nucleus were subjected to SDS-PAGE western blot analysis and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with the indicated primary antibodies at a dilution of 1:500 and HRP-conjugated secondary antibodies (dilution 1:2,000). The immune-reactive bands were visualized using a chemiluminescent substrate (Invitrogen) and were exposed to X-ray film.
6
Western Blot Analysis of Aβ Peptides in Bovine Brain Fractions
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Forty-seven samples (30 from frontal cortex, 10 from cerebellum, and 7 from brainstem) were obtained from 34 cattle. Sample buffer 3X was added to Tris, Triton, and SDS brain fractions, sonicated, and boiled for 5 min. Each sample and Aβ peptides 1–38, 1–40, and 1–42 were loaded onto urea gels and separated by electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare/Amersham Biosciences) using a Trans-Blot Semi Dry for 1 h. Membranes were boiled in a microwave for 3 min in TBS and incubated with primary monoclonal antibody (6E10 antibody, diluted 1:500, Covance) at 4°C overnight. After rinsing, membranes were incubated at RT for 30 min with AP-conjugated anti-mouse secondary antibody (diluted 1:12000, Invitrogen). Blots were developed using chemiluminescent substrate (Invitrogen) for 5 min and proteins were visualized on autoradiographic films.
7
Western Blot Protein Detection
8
PDGF Receptor Signaling in GIST-T1 Cells
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GIST-T1 cells were treated with CM from CAFscr, CAFshPDGFC #1, or CAFshPDGFC #2 for 24 h. The lysates with RIPA buffer (Cell Signaling Technology, Beverly, MA) were subjected to Western blot with the antibodies (1:1000 dilutions) against p-PDGFRA (Cell Signaling Technology, 4547P), PDGFRA (Cell Signaling Technology, 3164S), p-PDGFRB (ABclonal, Woburn, MA, Apo815), PDGFRB (Cell Signaling Technology, 3169 S), and β-actin (Cell Signaling Technology, 4967S). After SDS–polyacrylamide gel electrophoresis (Invitrogen) with the lysates, the gel was transferred to polyvinylidene difluoride membrane (Bio-Rad) with Trans-Blot Turbo transfer system 690BR (Bio-Rad). The immune-reactive bands with the secondary HRP-conjugated antibodies were visualized using a chemiluminescent substrate (Invitrogen) and were exposed to X-ray film (Genesee Scientific, San Diego, CA).
9
Western Blot Analysis of Protein Expression
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Cell lysates prepared with RIPA buffer were subjected to SDS–polyacrylamide gel electrophoresis (Invitrogen) and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with the primary antibodies (1:1000 dilutions). The antibodies used in the assay are in the Supplementary Table 1 . The immune-reactive bands were visualized using a chemiluminescent substrate (Invitrogen) and were exposed to X-ray film.
10
Western Blot Assay Protocol
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