Western blot analyses were performed using 50 μg of intestinal or renal protein extracts following earlier work [3] (link). For CaBP9k (anti-rabbit, Swant Swiss Antibodies, Switzerland), 4–20% Tris-HCl gels (BioRad, Hercules, CA) were used. For CYP27B1 (anti-rabbit, Santa Cruz Biotechnology Inc., CA) and CYP24A1 (Sigma Chemicals, MO), 12% Tris-HCl gels were used. All membranes were stripped and re-probed with β-actin antibody (anti-mouse, Chemicon International, MA). The membrane was blocked with 5% nonfat milk in Tris-buffered saline-Tween 20 (TBS-T) (0.1% Tween 20, 50 mmol/L Tris-HCL, 137 mmol/L NaCl, pH 7.4) buffer for 1 h. The blots were then incubated with the primary antibody, CaBP9k, CYP24A1 and CYP27B1 diluted 1∶1000, 1∶200 and 1∶1000, respectively, in 5% blocking agent in TBS-T buffer overnight at 4°C. Anti-β-actin antibody was diluted 1∶2000 in the same buffer but incubated with the membrane for one h at room temperature. The membranes were incubated with secondary antibodies, anti-rabbit (1∶20000, GE Healthcare Life Sciences, PA) or anti-mousse (1∶5000, GE Healthcare Life Sciences, PA), for one h at room temperature. The western blot was revealed by enhanced chemiluminescent (ECL) HRP substrate (Thermo Scientific, Rockford, IL USA).
Ecl hrp substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL HRP substrate is a chemiluminescent detection reagent used in Western blotting analysis. It is designed to generate a luminescent signal upon reaction with horseradish peroxidase (HRP), which is commonly used as a reporter enzyme in immunodetection assays. The substrate provides a sensitive and reliable method for the detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Tris hcl gel, by Bio-Rad (1 mentions)
β actin antibody, by Merck Group (1 mentions)
Cyp27b1, by Santa Cruz Biotechnology (1 mentions)
Cyp24a1, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation assay (ripa), by Solarbio (1 mentions)
Mouse anti rabv g monoclonal antibody, by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l, by Proteintech (1 mentions)
Anti mouse horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Uvp biospectrum imaging system, by Analytik Jena (1 mentions)
Cd63 sc 5275, by Santa Cruz Biotechnology (1 mentions)
Gapdh sc 20357, by Santa Cruz Biotechnology (1 mentions)
Alix sc 53540, by Santa Cruz Biotechnology (1 mentions)
Hsp70 sc 24, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Clone ac 74, by Merck Group (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
8 protocols using ecl hrp substrate
1
Quantifying Protein Expression in Tissues
2
AAV9-Mediated RABV G Protein Expression in 293T Cells
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The 293T cells in a 6-well plate were infected with AAV9-RABVG or AAV9-GFP at a multiple of infection (MOI) of 105, respectively. The cells inoculated with DMEM were the mock cells. As for IFA, the cells were fixed with 80% ice-cold acetone for 12 h at −20°C after being cultured for 72 h at 37°C, and then stained with primary antibody of mouse anti-RABV G monoclonal antibody (Millipore, USA) and second antibody of Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L) (Proteintech, China). The fluorescent signals were observed under an inverted fluorescence microscope (Olympus, IX71, Japan). As for immunoblot, the 293T cells infected with AAV9-RABVG, AAV9-GFP, or mock, respectively, were lysed in 1 ml RIPA (Solarbio, China) per 106 cells with 0.5 mM proteinase inhibition of Phenylmethyl sulphonyl Fluoride (PMSF). The supernatants of cell lysates were separated by a 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane (Millipore, USA). The membrane was incubated with primary antibody of mouse anti-RABV G monoclonal antibody (Millipore, USA) and secondary antibody of donkey anti-mouse IgG H&L (HRP) (Abcam, UK), and visualized by the enhanced chemiluminescent (ECL) HRP substrate (Thermo, USA).
3
Western Blot Analysis of Exosomal Markers
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Equal sample volumes (16 μL) were lysed with 4 μL of 5X reducing sample buffer (1 M Tris-HCl (pH 6.8), 30% glycerol, 6% SDS, 3% 2-mercaptoethanol, 0.005% bromophenol blue) and boiled at 95°C for 5 min. Pooled samples (n = 12) were separated onto a 10% SDS-PAGE and electro-transferred onto a nitrocellulose membrane (Amersham, UK). Membranes were blocked with 5% non-fat skimmed milk in TBST, followed by overnight incubation with the following primary exosomes specific antibodies diluted at 1:500 HSP70 (sc-24, Santa Cruz, TX, USA), CD63 (Sc-5275, Santa Cruz), GM130 (P-20, sc-16268, Santa Cruz), Alix (sc-53540, Santa Cruz), and GAPDH (sc-20357, Santa Cruz). Subsequently, horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody (Sigma Aldrich) diluted at 1:2500 was applied for 1h. Finally, membranes were washed thrice with TBST, and the enhanced chemiluminescent (ECL) HRP substrate (Thermo Scientific, Waltham, USA) was used for signal development. Chemiluminescence was detected using the UVP Biospectrum Imaging System and Image Acquisition and Analysis Software (Analytik Jena, Cambridge, UK).
4
Colon Protein Analysis by Western Blot
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Western Immunoblot analysis was performed on colon lysates extracted by mechanical homogenisation in RIPA buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). Samples were clarified, denatured in sample buffer and boiled for 5 min. Protein lysate was fractionated on 10% SDS-PAGE, transferred to polyvinylidene fluoride membranes (Millipore) and probed with primary antibody to IL-10 (1:500 dilution, Proteintech, Rosemont, IL, USA) or murine β-actin (1:1000 dilution, Clone AC-74, Sigma), incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and visualised using ECL HRP substrate (ThermoFisher, Waltham, MA, USA), GelAS 4000 and ImageLab software.
5
Protein Elution and Visualization
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To elute bound protein, SpyC-antigen coated beads were re-suspended in 1x LDS buffer (NuPAGE; Life Technologies, #NP0007) with 10% reducing agent (NuPAGE; Life Technologies, #NP0009) and boiled at 95 °C for 10 min. The beads were pelleted by centrifugation and the eluted protein fraction was separated on a Novex 4–12% Bis-Tris Gel (Life Technologies, #NP0321) for 38 min at 200 V. Separated protein fractions were visualized by Coomassie Blue staining (InstantBlue; Expedeon, #ISB1L) or transferred to nitrocellulose membranes (iBlot2; Thermo Fisher Scientific, #IB23001). After blocking with 5% milk/PBST, the membranes were incubated with an HRP-conjugated anti-HA tag antibody (Thermo Fisher Scientific, #PA1-29751), diluted 1:1000 in PBS containing 1% BSA. Membranes were washed four times in PBST and HA-tagged protein was detected with an enhanced chemi-luminescent (ECL) HRP substrate (Thermo Fisher Scientific, #34080), followed by image acquisition using a Fusion FX imaging system (Vilber).
6
Autophagic Flux Measurement via LC3B
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To measure autophagic flux, cellular LC3B was detected by western blot analysis. HEK293T cells were seeded in 6-well plates and transfected with plasmids encoding either BaTdp or BaTdp (G164A) using Turbofect reagent. Total plasmid concentration was normalised to 1 μg/mL growth medium using empty pCDNA vector. At 24 and 48 hours post-transfection, cells were harvested and lysed by boiling in PBS supplemented with 1% SDS. Lysates were resolved by SDS-PAGE and analysed for LC3B content by Western blot with bands visualised using enhanced chemiluminescent (ECL) HRP substrate (Thermo Scientific) and detected with an ImageQuant LAS 4000 mini system (GE Healthcare).
7
Western Blot Analysis of Autophagy Markers
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Lysates were prepared from tumor cells and analyzed by western blotting. Primary antibodies include FIP200 (1:1000, Cell Signaling #12436), p62 (1:2000, Cell Signaling #39749), TSC1 (1:1000, Cell Signaling #6935), phospho-ULK1 (Ser757) (1:1000, Cell Signaling #6888), ULK1 (1:1000, Cell Signaling #8054), LC3B (1:2000, Cell Signaling #43566), phospho-p70 S6K (Thr389) (1:1000, Cell Signaling #9205), p70 S6K (1:1000, Santa Cruz #sc-230), Atg5 (1:1000, Cell Signaling #12994), Atg7 (1:1000, Cell Signaling #8558), phospho-Stat3 Y705 (1:1000, Cell Signaling #9145), Stat3 (1:1000, Cell Signaling #3139), Osteopontin (1:1000, R&D Systems #MAB808), GAPDH (1:5000, Cell Signaling #5174), β-actin (1:10000, Sigma-Aldrich #), Vinculin (1:10000, Sigma-Aldrich #V4505) and HRP-linked anti-Rabbit IgG or anti-Mouse IgG (1:10000, Cell Signaling) were used as secondary antibodies. ECL HRP substrate (Thermo Scientific) was used for signal imaging.
8
Quantitative Analysis of ST6GalNAc5 and ST6Gal1 in Pancreatic Cancer
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The primary antibodies, rabbit polyclonal antibodies against ST6GalNAc5 (GTX45949), ST6Gal1 (GTX104018) and β-actin (GTX109369), and secondary HRP-conjugated goat anti-rabbit antibody were purchased from GeneTex. Each 100 μg of total lysate proteins from PANC-1 CCs or CSCs were separated by SDS PAGE before transferred onto a PVDF membrane. The primary antibodies against ST6GalNAc5, ST6Gal1 and β-actin were diluted by 1000, 1000 and 5000 folds, respectively, in blocking solution (20 mM Tris-HCl, pH 7.5/150 mM NaCl/3% non-fat milk). After incubation with the primary antibodies for 2 hours, the membranes were washed with wash solution (20 mM Tris-HCl, pH 7.5/150 mM NaCl/0.05% Tween-20) for 4 times. Then the membranes were incubated with the secondary antibody which was diluted in blocking solution for 2500 folds for 2 hours and washed for 4 times. ECL HRP substrate (Thermo-Fisher, 34075) was used to perform the chemiluminescent signals and images were taken with a CCD imaging system.
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