Detection of Dnaic1 protein by Western blotting was performed using a mouse monoclonal antibody generated against a synthetic peptide from the human DNAI1 protein 18 (link) using standard procedures. For analysis of mouse tracheal epithelial cells, total cell lysates were prepared in Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Ciliary axonemes were isolated from cultured mouse tracheal epithelial cells as previously described 32 (link) and prepared in gel-loading buffer. For studies of protein turnover, both the soluble (cytoplasmic) and pellet (axonemal) fractions from individual cultures were analyzed. Protein samples were fractionated on 4 to 12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose membranes, and probed using Amersham ECL Plus reagents (GE Healthcare, Buckinghamshire, UK), all according to manufacturer's instructions. For normalization of loading, Western blots of cytoplasmic extracts were reprobed with an antibody to GAPDH (Sigma, St. Louis, MO), and axonemal pellets were reprobed with mouse anti-acetylated α-tubulin (Invitrogen). Quantification of signals was performed on an Odyssey Imaging System (Licor, Lincoln, NB).
Amersham ecl plus reagent
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
Amersham ECL PLUS reagent is a chemiluminescent detection system for the identification and quantification of proteins in Western blot analysis. The reagent produces a light-emitting reaction when combined with the target protein, which can be detected and measured using a compatible imager or film.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (2 mentions)
4 to 12 bis tris gradient gels, by Thermo Fisher Scientific (2 mentions)
An antibody to gapdh, by Merck Group (2 mentions)
Mouse anti acetylated α tubulin, by Thermo Fisher Scientific (2 mentions)
Anti mouse hrp conjugated antibody, by Bio-Rad (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
β actin, by BD (2 mentions)
β actin, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Amersham hybond ecl membrane, by GE Healthcare (1 mentions)
Bio safe coomassie stain, by Bio-Rad (1 mentions)
C digit scanner, by LI COR (1 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Py705 stat3, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (1 mentions)
Cox 2, by Cayman Chemical (1 mentions)
P sapk jnk, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
12 protocols using amersham ecl plus reagent
1
DNAIC1 Protein Detection in Mouse Trachea
2
DNAIC1 Protein Detection in Mouse Trachea
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Detection of Dnaic1 protein by Western blotting was performed using a mouse monoclonal antibody generated against a synthetic peptide from the human DNAI1 protein 18 (link) using standard procedures. For analysis of mouse tracheal epithelial cells, total cell lysates were prepared in Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Ciliary axonemes were isolated from cultured mouse tracheal epithelial cells as previously described 32 (link) and prepared in gel-loading buffer. For studies of protein turnover, both the soluble (cytoplasmic) and pellet (axonemal) fractions from individual cultures were analyzed. Protein samples were fractionated on 4 to 12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose membranes, and probed using Amersham ECL Plus reagents (GE Healthcare, Buckinghamshire, UK), all according to manufacturer's instructions. For normalization of loading, Western blots of cytoplasmic extracts were reprobed with an antibody to GAPDH (Sigma, St. Louis, MO), and axonemal pellets were reprobed with mouse anti-acetylated α-tubulin (Invitrogen). Quantification of signals was performed on an Odyssey Imaging System (Licor, Lincoln, NB).
3
Quantification of COMT Protein in Mouse Brain
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The striatum and cerebellum were dissected from COMT-OE and control littermate mice. Tissue was homogenized in lysis buffer (25 mM Tris pH 8.0, 125 mM NaCl, 1% Triton, with complete protease inhibitor cocktail [Roche]). Samples were centrifuged at 16 kg for 30 min at 4°C and the supernatant collected. A Biorad protein assay kit (Biorad) was used to determine protein concentration and 10-µg total protein was loaded in each lane of a 15% SDS Page gel. After transfer to Immobilon-FL PDVF membrane (Millipore) the blots were incubated with a purified mouse monoclonal anti-mouse catechol-O-methyltransferase antibody (BD Transduction Laboratories) 1:5000, then an HRP conjugated goat anti-mouse Ig antibody (1:5000). Amersham ECL Plus reagents (GE Healthcare Life Sciences) were used for detection. Following detection, blots were stripped and reprobed for tubulin as a loading control.
4
Osteogenic Gene Expression in MC3T3 Cells
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For the determination of osteogenic-associated gene expression level of MC3T3 cells which were cocultured with different nanofiber mats, MC3T3 cells (1 × 105 cells/well) were cultured in a 6-well plate for 1, 4, and 7 days. At a period of 1, 4, and 7 days of culture, sodium dodecyl sulfate polyacrylamide gel electrophoresis was used for the protein extraction, which was then moved onto a polyvinylidene difluoride membrane surface (Merck Millipore, German). The membranes were then treated with 5% bovine serum albumin (BSA) and then incubated with monoclonal antibodies for RUNX2, OCN, and actin at 4°C overnight. The samples were then incubated with HRP-conjugated secondary goat anti-rabbit antibody (RT, 2 h), followed by the quantitative analysis using Amersham ECL Plus reagents on an Image Quant LAS 4000 (GE, United States).
5
SDS-PAGE and Western Blot Analysis of T4P
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Purified T4P were separated by SDS-PAGE using 15% polyacrylamide gels. Gels were stained using Bio-Safe Coomassie stain (BioRad) or blotted to Amersham Hybond ECL membranes (GE Healthcare) using standard molecular biology techniques52 . Blocking, incubation with primary/secondary antibodies and detection using Amersham ECL Plus reagents (GE Healthcare) were done following the manufacturer’s instructions. The primary antibody was a previously described rabbit anti-PilE serum (1/2500)59 (link), while the secondary antibody (1/10,000) was a commercial Amersham ECL anti-rabbit IgG HRP-linked whole antibody (GE Healthcare). Full scan Coomassie-stained gels and/or immunoblots can be seen in the Source Data file.
6
Western Blot Analysis of SH2D1a Protein
7
Western Blot Analysis of SH2D1a Protein
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Total cell protein extracts were prepared from PBMCs stimulated with Mtb-Ag for 5 days. In different experiments, PBMCs stimulated with Mtb-Ag for 5 days were exhaustively washed and then cultured in complete RPMI-1640 medium supplemented with 100 U/mL of rhIL-2 for at least 7 days before obtaining total cell protein extracts. Western blotting was performed by standard methods. Each nitrocellulose membrane was blotted with mouse monoclonal antibodies to SH2D1a (SAP, cat. 14-9888-82, eBioscience), stripped, and reblotted with GAPDH (Ambion) or β-actin (BD Biosciences). Bound antibodies were detected with an anti-mouse HRP-conjugated antibody (BioRad, Hercules, CA, USA) using Amersham ECL PLUS reagent (GE Healthcare). Images were obtained with an Intelligent Dark Box (Fujifilm LAS1000) and analyzed with ImageJ Analysis software (NIH, Bethesda, MA, USA). The intensity of each band was expressed as arbitrary units (AU).
8
Western Blot Analysis of DWV Antigens
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Protein from total pupa homogenate was precipitated by the addition of 4 volumes of ice-cold acetone for Western blot analysis. The pellet was resuspended in distilled water and boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer. Samples were separated on 7.5% acrylamide gels prior to electrophoretic transfer to nitrocellulose membranes (Pall corporation, Port Washington, New York). To detect DWV antigens, blots were blocked with 5% skim milk powder and probed with different mouse monoclonal antibodies against DWV VP1. The blots were developed using Amersham ECL plus reagent (GE Healthcare, Chalfont St Giles, GB) and exposed to a C-Digit scanner (Licor, Lincoln, Nebraska) and X-ray films for imaging.
9
Western Blot Analysis of Cellular Proteins
10
Western Blot Analysis of Insulin Signaling
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Muscle and liver samples were lysed, and the proteins were separated by SDS polyacrylamide gel electrophoresis on 4–12% Criterion XT gels (BioRad) and then transferred to nitrocellulose membranes (Schleicher & Schuell). After blocking, the membranes were probed with mouse antibodies diluted 1/1000 in milk. The antibodies used were against: p(Ser473)Akt, Akt, p(Ser536)NFκB p65, p(Ser176/180)IKKα/β p(Ser9)GSK-3β and β-actin (Cell Signaling Technology). Antibody binding was revealed with a peroxydase-coupled secondary antibody diluted in milk, detected using the Amersham ECL Plus reagent (GE Healthcare) and quantified by Image Quant TL software (GE Healthcare Bio-sciences). The membranes were stripped and re-probed with the anti-β-actin antibody as a control for loading. Insulin signaling molecule phosphorylation was performed in stimulated state after an euglycemic hyperinsulinemic clamp procedure.
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