FimA levels were determined as described earlier (61 (link)). Strains were cultured in LB with or without 0.5-mM CuSO4 static for 24 h. OD600 of overnight cultures were adjusted to 1.0, acidified with water (pH 1.8), and denatured. An equal amount of protein was separated on 15% SDS-PAGE and transferred to 0.45-μm PVDF membrane (Millipore). Blots were blocked with 5% skim milk, probed with FimA antibody (1:10,000 dilution), detected with anti-rabbit IgG secondary antibody (1:10,000 dilution, Invitrogen), and visualized with ECL-Prime reagents (Amersham). Images were acquired in a Chemidoc system (Biorad). Duplicate gels were stained with Commassie blue to serve as loading controls.
Ecl prime reagent
Manufactured by Cytiva
Sourced in United Kingdom, Japan
ECL Prime reagent is a chemiluminescent detection solution used for western blotting. It produces a stable light signal for the detection of proteins on membranes.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 500, by Cytiva (3 mentions)
Nitrocellulose membrane, by Cytiva (3 mentions)
Amersham imagequant 800 imager, by Cytiva (3 mentions)
Hrp conjugated anti rabbit igg, by Cytiva (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Supersep ace 15 precast gel, by Fujifilm (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Chemidoc mp imager, by Bio-Rad (2 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
0.45 μm pvdf membrane, by Merck Group (1 mentions)
Anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Tricine sample buffer, by Bio-Rad (1 mentions)
Gelsolin, by Abcam (1 mentions)
Protran nitrocellulose membrane, by Cytiva (1 mentions)
Peroxidase conjugated anti mouse or anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Lamin a c, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Cell lysis buffer, by Thermo Fisher Scientific (1 mentions)
36 protocols using ecl prime reagent
1
Quantifying FimA Protein Levels
2
Immunoblot Analysis of PRL-3 in HEK293 Cells
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HEK293 stable cell lines
were lysed in standard HNTG lysis buffer, and cleared lysate was normalized
to total protein amount and subjected to sodium dodecylsulfate-polyacrylamide
gel electrophoresis and immunoblot analysis using anti-PRL-3-specific
monoclonal antibody (mouse, EMBL in-house antibody facility, clone
1E7, used 1:1000). Blocking was performed in TBS-Tween (0.1% (v/v))
containing 5% (w/v) BSA, according to standard procedures. Anti-tubulin-specific
antibody (mouse, Sigma, clone T-5168, used 1:5000) was used according
to the manufacturer’s instructions. Detection of respective
peroxidase-coupled secondary antibody (Sigma or GE Healthcare, used
1:10 000) was carried out with ECL prime reagents from Amersham.
were lysed in standard HNTG lysis buffer, and cleared lysate was normalized
to total protein amount and subjected to sodium dodecylsulfate-polyacrylamide
gel electrophoresis and immunoblot analysis using anti-PRL-3-specific
monoclonal antibody (mouse, EMBL in-house antibody facility, clone
1E7, used 1:1000). Blocking was performed in TBS-Tween (0.1% (v/v))
containing 5% (w/v) BSA, according to standard procedures. Anti-tubulin-specific
antibody (mouse, Sigma, clone T-5168, used 1:5000) was used according
to the manufacturer’s instructions. Detection of respective
peroxidase-coupled secondary antibody (Sigma or GE Healthcare, used
1:10 000) was carried out with ECL prime reagents from Amersham.
3
Western Blot Analysis of Protein Expression
4
Cell Growth and Protein Expression Analysis
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Cell number was evaluated via the Hoechst assay, after 1, 7, and 14 days [10 (link)]. Constructs were digested at 60 °C in papain buffer [11 (link)]. Briefly, 10 µl of papain digest was added to Hoechst dye (Bisbenzimide H) for a final dye concentration of 0.1 µg/ml and fluorescence was measured (360 nm Ex, 460 nm Em). Metabolic activity was measured using an alamarBlue® assay (Invitrogen, UK) over 14 days. Samples were incubated for 4 h (37 °C) in 10 % alamarBlue® and fluorescence was read.
Protein expression at 14 days was analyzed using SDS-PAGE and Western blotting, as in Hernandez et al. [12 (link)]. Scaffolds were incubated in cell lysis buffer (Invitrogen) and mixed with sodium dodecyl-sulphate (SDS) sample buffer. Standards were prepared from human cartilage, snap frozen in liquid nitrogen, milled using a dismembrator (B Braun Biotech International GmbH), and suspended in cell lysis buffer (100 mg/ml). Scaffolds were probed for fibronectin (SC-9068, Santa Cruz Biotech), then stripped and reprobed for GAPDH as a control. Membranes were developed using ECL prime reagents (Amersham) and signal strength assessed using a densitometer.
Protein expression at 14 days was analyzed using SDS-PAGE and Western blotting, as in Hernandez et al. [12 (link)]. Scaffolds were incubated in cell lysis buffer (Invitrogen) and mixed with sodium dodecyl-sulphate (SDS) sample buffer. Standards were prepared from human cartilage, snap frozen in liquid nitrogen, milled using a dismembrator (B Braun Biotech International GmbH), and suspended in cell lysis buffer (100 mg/ml). Scaffolds were probed for fibronectin (SC-9068, Santa Cruz Biotech), then stripped and reprobed for GAPDH as a control. Membranes were developed using ECL prime reagents (Amersham) and signal strength assessed using a densitometer.
5
Investigating MEK and Akt Signaling
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Reagents. Dulbecco's modified Eagle's medium (DMEM) was purchased from Thermo Scientific (hemel hempstead, UK). Fetal bovine serum (FBS) was purchased from HyClone Laboratories (Logan, UT, USA). Phenol red-free DMEM and penicillin (100 U/ml) and 100 mg/ml streptomycin were purchased from Life Technologies (Rockville, MD, USA). UO126 was purchased from Selleck Chemicals (Houston, TX, USA). SB253580 and LY294002 were purchased from Tocris Bioscience (Ellisville, MO, USA). 4-Hydroxytamoxifen (4-OHT) was purchased from Sigma (St. Louis, MO, USA). The secondary horseradish peroxidase (HRP)-conjugated and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Rabbit polyclonal anti-MEK and anti-Akt antibodies (total and phospho-form) were purchased from Cell Signaling Technology (Beverly, MA, USA). Recombinant human IL-8 was purchased from R&D Systems (Minneapolis, MN, USA). The ECL prime reagents were purchased from Amersham (Buckinghamshire, UK).
6
SARS-CoV-2 Nucleocapsid Protein Detection
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Recombinant proteins and cell lysate proteins were detected using Western blotting. Before harvesting, MH7A cells and FLS were washed with PBS. The cells were homogenized in 2% sodium dodecyl sulfate sample buffer using BioMasher II (Nippi Inc., Tokyo, Japan). The samples were then centrifuged for 5 min at 15,000× g and 20–25 °C. Proteins were processed using a SuperSep Ace 10% precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred onto a polyvinylidene fluoride membrane. The membranes were probed with anti-β-actin (mouse monoclonal, clone AC-15; Sigma-Aldrich), anti-NRP1 (rabbit monoclonal, clone EPR3113; Abcam), and anti-NRP2 (rabbit polyclonal; Sigma-Aldrich) antibodies. Rabbit sera against anti-nucleocapsid (N) protein were prepared by immunizing rabbits with the N-terminal domain of SARS-CoV-2 N protein expressed and purified in E. coli. Horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) were then added. Horseradish peroxidase activity was detected using ECL prime reagents (Cytiva, Tokyo, Japan), followed by imaging using an Image Quant LAS 500 (Cytiva) system.
7
Drosophila Embryo Immunoblotting Protocol
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Drosophila embryo extracts were generated for immunoblotting as described (McClintock et al, 2018 (link)). SDS-PAGE and protein transfer to polyvinylidene difluoride (PVDF) membrane (Immobilon-P, Merck Millipore) was performed with the NuPAGE Novex and XCell II Blot Module systems (ThermoFisher Scientific) according to the manufacturer’s instructions. Membranes were blocked with 5% dried skimmed milk powder (Marvel) and washed in PBS/0.05% Tween-20. Details of primary and secondary antibodies, including working dilutions, are provided in Table S5 and S6 . Secondary antibodies were detected with ECL Prime reagents (Cytiva Amersham) as instructed by the manufacturer.
8
Western Blotting of GFP-labeled Proteins
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The plated cells were washed with phosphate-buffered saline before collection. Proteins from the cultured cells were processed using SuperSep Ace 15% precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred on to a polyvinylidene fluoride membrane. The membranes were probed with anti-GFP (0.5 µg/mL, chicken polyclonal; Genscript, Piscataway, NJ, USA) and anti-β-actin (1 µg/mL, mouse monoclonal, clone. AC-15; Sigma-Aldrich) antibodies. Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were then added. Horseradish peroxidase activity was detected using ECL prime reagents (Cytiva, Tokyo, Japan), followed by imaging with Image Quant LAS 500 (Cytiva).
9
Harvesting and Quantifying Intracellular and Secreted Proteins
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Proteins were harvested from the intracellular and culture supernatants. Before collection, MH7A cells, FLS, monocytes, and macrophages were washed with phosphate-buffered saline. The cells were dissolved in radioimmunoprecipitation assay (RIPA) buffer. For the supernatant protein assay, the last 24 h of incubation was performed in a serum-free medium. Cell supernatants for FLS, MH7A, HEK293T, and macrophages were suspended in acetone and kept at –20 °C for 1 h. The samples were then centrifuged for 10 min at 15,000 × g at 20–25°C. Protein precipitates were dried for 30 min and dissolved in RIPA buffer. Proteins were processed using a SuperSep Ace 15 % precast gel (FUJIFILM Wako Pure Chemical Co.) and transferred onto a polyvinylidene fluoride membrane. The membranes were probed with anti-human β-actin antibody (0.1 μg/mL, mouse monoclonal, clone AC-15, Sigma-Aldrich), anti-human FXIII-A antibody (0.025 μg/mL, mouse monoclonal, Proteintech), anti-human FXIII-B antibody (0.1 μg/mL, rabbit polyclonal, Sigma-Aldrich), and anti-HA-tag antibody (0.2 μg/mL, goat polyclonal, Genscript). Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) were then added. Horseradish peroxidase activity was detected using ECL prime reagents (Cytiva, Tokyo, Japan), followed by imaging using the Image Quant LAS 500 (Cytiva) system.
10
TRPV1 Protein Expression Analysis by Immunoblotting
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