Proteins were transferred onto polyvinylidene difluoride membranes for immunodetection using electrophoretic semi-dry transfer system. After transfer, membranes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature and incubated with specific primary antibody (indicated above, dilution 1:1,000 in 1.5% BSA/PBS-T) overnight at 4 °C. Membranes were washed in PBS-T before addition of HRP-coupled secondary antibodies (indicated above, 1:7,000 in 0.75% BSA in PBS-T; 1 h, room temperature). HRP was detected by enhanced chemiluminescence using the LumiGlo reagent (Cell Signaling) according to the manufacturer's instructions. Images have been cropped for presentation. Full-size images are presented in Supplementary Fig. 7 .
Lumiglo reagent
Manufactured by Cell Signaling Technology
Sourced in United States, Germany, Italy, United Kingdom
LumiGLO reagent is a chemiluminescent substrate used for the detection of immobilized proteins in Western blotting applications. It is designed to produce a luminescent signal when exposed to horseradish peroxidase (HRP), which is commonly used as a reporter enzyme in immunodetection procedures.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (5 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Chemidoc xrs gel documentation system, by Bio-Rad (4 mentions)
Pvdf membrane, by Roche (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Peroxide detection system, by Cell Signaling Technology (3 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Anti hif 1α, by BD (2 mentions)
Anti pfkfb4 ab137785, by Abcam (2 mentions)
Anti phosphor rb ser795, by Cell Signaling Technology (2 mentions)
Anti cdk6, by Cell Signaling Technology (2 mentions)
Chemidoc xrs station, by Bio-Rad (2 mentions)
Criterion tgx, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Tris acetate gel, by Bio-Rad (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Mouse anti atg5 7c6, by Nanotools (2 mentions)
67 protocols using lumiglo reagent
1
Western Blot Immunodetection Protocol
2
Western Blot Analysis of ZIP7 Phosphorylation
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Cells were lysed by sonication using a Vibra Cell sonicator (Sonics & Materials, USA) and heated for 5 min at 95 °C. An equivalent of 3x106 cells per lane was separated at 170 V on 10% polyacrylamide gels. After separation, samples were blotted onto nitrocellulose membranes (GE Healthcare Life Sciences, Germany). Uniform loading of gels was confirmed by Ponceau S (AppliChem, Germany) staining. Subsequently, membranes were blocked for 1 h with TBS-T (20 mM Tris–HCl [pH 7.6], 137 mM NaCl, 0.1% [v/v] Tween 20), containing 5% fat-free dry milk, and were washed afterwards thrice with TBS-T. Incubation with primary mouse monoclonal pZIP7 (S275/S276) [21] (link), ZIP7 (ProteinTech, United Kingdom) and β-actin (Cell Signaling Technology, Germany) antibodies was performed overnight at 4 °C at 1/1000 dilution in TBS-T, containing 5% BSA. Afterwards, membranes were washed thrice with TBS-T and incubated with either anti-rabbit-HRP (for β-actin and ZIP7) or anti-mouse-HRP (for pZIP7) (both from Cell Signaling Technology, Germany) secondary antibodies 1/2000 in TBS-T with 5% fat-free dry milk. After again washing thrice with TBS-T, immunodetection was performed using LumiGlo Reagent (Cell Signaling Technology, Germany) on LAS-3000 (Fujifilm Lifescience, Germany). Band density was determined with ImageJ software (NIH, USA).
3
Immunoblotting of Cellular Proteins
4
Western Blot Analysis of Neprilysin and GAPDH
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Cultured cells or tissues were lysed with sodium dodecyl sulfate (SDS) sample buffer and boiled for 10 min. The protein content was normalized using protein assay kits (Bio-Rad, Hercules, CA). Equal amounts of total protein were separated by SDS polyacrylamide gel electrophoresis, electrophoretic transferred to a polyvinylidene-fluoride membrane, and immunoblotted with primary antibodies (Neprilysin/CD10 antibody, NBP2-15771, Novus Biologicals, Littleton, CO; GAPDH polyclonal antibody, 2172, 10494-1-AP, Proteintech, Hubei, China) overnight. After washing three times using tris-buffered saline with Tween (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h. After washing thrice with TBST, the secondary antibodies were visualized using the LumiGLO Reagent (Cell Signaling Technology, Danvers, MA).
5
Whole-cell Lysate Protein Analysis
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The whole-cell lysate of L. monocytogenes F4244, P. aeruginosa PRI99, E. coli EDL933, and S. Enteritidis PT21 overnight cultures (5 mL each) was prepared by sonication (Branson, Danbury, CT, United States). Bacterial samples were sonicated in an ice bucket (three 10 s cycles at 30-s intervals) and centrifuged for 10 min at 14,000 rpm (Eppendorf) at 4°C to separate the soluble fraction (supernatant) from the bacterial debris (pellet). The protein concentration was determined by the BCA method (Thermo Fisher Scientific). Equal amounts of proteins were separated on SDS-PAGE gel (10% polyacrylamide) and electro-transferred to polyvinylidene difluoride (PVDF) membrane (Fisher Scientific) (Singh et al., 2016 (link); Drolia et al., 2018 (link)). Primary and secondary antibodies were diluted as above. Membranes were first probed with mAb-2F11 at 4°C overnight, and then with anti-mouse HRP conjugated antibody at room temperature for 1.5 h. LumiGLO reagent (Cell-Signaling Technology) was used to visualize the bands using the Chemi-Doc XRS system (Bio-Rad).
6
Protein Extraction and Western Blotting
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Tissues or cells were lysed in radioimmunoprecipitation assay buffer containing 0.5% cholic acid (Sigma-Aldrich, C1129), 0.1% sodium dodecyl sulfate (Sigma-Aldrich, L3771), 2 mM ethylenediaminetetraacetic acid, 1% Triton X-100 (Sigma-Aldrich, T8787), 10% glycerol (Sigma-Aldrich, G5516), 1.0 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, 78830), 1 µg/mL aprotinin (Sigma-Aldrich, 10236624001). After sonication for 30 s on ice and centrifuging for 20 min at 13,000 g at 4°C, the supernatant was collected, and protein concentration was determined by protein assay kit (Bio-Rad, 5000122), using bovine serum albumin (BSA; Bio-Rad, 5000122) as the standard. Protein samples (20 µg) were resolved by SDS-PAGE. Proteins were blotted onto nitrocellulose membranes (Bio-Rad, 1620177). Non-specific binding was blocked in 5% blocking buffer (5% non-fat dry milk, 150 mmol/L Tris-HCl, pH 7.4, 50 mmol/L NaCl, 0.05% Tween 20 [Sigma-Aldrich, P9416]) for 1 h, and membranes were incubated overnight at 4°C with the indicated with primary antibody at a 1:1000 dilution. The membranes were then incubated for 1 h with horseradish peroxidase-coupled secondary antibody at room temperature. Chemiluminescent signals were then developed with LumiGLO reagent (Cell Signaling Technology, 7003S) and exposed on X-ray film (Fuji Photo Film Co., Ltd.).
7
Western Blot Analysis of HIF-1α, PFKFB4, and Rb
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Cells were lysed with cell lysis buffer (#9803, Cell Signaling Technology, Inc., USA), and equal amounts of cell lysates were electrophoretically separated using 8%, 10%, or 12% SDS-PAGE gels. The proteins were transferred to a PVDF membrane (Roche, USA). The membrane was blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature and then incubated overnight at 4°C with antibodies. The following antibodies were used: anti-HIF-1α (#61959) from BD Transduction Laboratories (USA); anti-PFKFB4 (ab137785) from Abcam (USA); and anti-phosphor-Rb (Ser795) (#9301) and anti-CDK6 (#3136) from Cell Signaling Technology, Inc. (USA). Anti-beta-actin (#3700, Cell Signaling Technology, Inc., USA) was used as a loading control. HRP-conjugated anti-rabbit or anti-mouse (Santa Cruz) secondary antibodies were used. All blots were developed with 20× LumiGLO® Reagent and 20× peroxide (#7003, Cell Signaling Technology, Inc., USA).
8
NF-κB2 and ERK1/2 Activation in B Cells
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Flow cytometry sorted B cells were plated at 2x106 cells/ml in 96-well flat bottom plates. Cells were stimulated in RPMI 1640 complete medium alone or with increasing concentrations of BAFF. After 24 hours of stimulation, the processing of NF-κB2 p100 and phosphorylation of ERK1/2 was determined by Western blot analysis. Cells were first washed with PBS and then lysed in 1X NuPAGE LDS sample buffer supplemented with NuPAGE sample reducing agent (Invitrogen). Lysates were separated on 4–20% Mini-PROTEAN TGX gels (BioRad) and transferred to nitrocellulose using the iBlot transfer system (Invitrogen). The membranes were probed using anti- NF-κB2 p100/p52 or anti-Phospho ERK1/2 (T202/Y204). Goat anti-rabbit IgG-HRP were used to detect antibody binding (Cell Signaling Technology). Blots were then stripped and probed with anti-β actin-HRP or anti-ERK1/2-HRP antibodies (Cell Signaling Technology). Blots were developed using LumiGLO reagent (Cell Signaling Technology) and visualized using Innotech FluorChem Imaging System (Protein Simple, Santa Clare, CA).
9
Western Blot Analysis of Phospho-ERK1/2
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Cells were lysed in 50 mM Tris HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% (v/v) NP40, 5 mM NaF, 0.25% deoxycholate, and 2 mM NaVO3, and samples were separated on a 12% polyacrylamide gel. Proteins were transferred to a PVDF membrane (Millipore) and probed with either anti-phospho-ERK1/2 (Cell Signaling) or anti-ERK1/2 (Cell Signaling) antibodies. All secondary antibodies were horse radish peroxidase conjugated, and membranes were developed with LumiGLO reagent (Cell Signaling). Uncropped western blot images are shown in Supplementary Fig. 14 .
10
Quantifying Extracellular GRP78 in Multiple Myeloma
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Conditioned supernatants of multiple myeloma NCI-H929 cells were obtained by washing the cells in PBS and culturing them in Protein-Free Hybridoma medium for 72 h. Supernatants were collected and centrifuged at 12,000 × g for 30 min to remove cell debris. They were further concentrated using Amicon Ultra-0.5 centrifugal filter unit with ultracel-10 membrane (UFC501096, Milipore). For dot blot assay, 1 μl aliquots of 5 x concentrated samples were spotted on nitrocellulose membranes (Protean BA 85, GE Healthcare Life Sciences, Whatman). Released proteins were denatured, separated with 4–20% SDS-PAGE (Criterion TGX, Bio-Rad) and transferred to an Immuno-Blot TM polyvinylidene difluoride (PVDF) membrane (Bio-Rad) in western blot analysis. After blocking the membrane in 3% BSA dissolved in TBS, membranes were incubated 2 h at room temperature in 3% BSA with a primary polyclonal antibody GRP78 supplied with the GRP78-ELISA Kit (Biovendor). Afterwards, membranes were incubated with an anti-biotin HRP conjugated antibody (CST) diluted 1:1000. After washing, a chemiluminescent substrate (LumiGLO Reagent and Peroxide, Cell Signaling Technology) was added to the membrane, which was then exposed in the Chemidoc XRS station (Bio-Rad Laboratories).
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