Whole cell lysates were collected from the indicated cell lines, separated by SDS-PAGE, transferred onto nitrocellulose membranes (BioRad), blocked with 5% milk for 1 h at room temperature, and probed with the following primary antibodies overnight at 4°C: rabbit anti-HJURP (generated against a C-terminal fragment; 1 μg/mL; Barnhart et al., 2011 (link)), human ACA (Antibodies Incorporated 15-235, 1:500), and mouse monoclonal anti-α-tubulin (Sigma-Aldrich T9026, 1:4000). The next day, blots were washed 3x in PBST and probed with the following secondary antibodies for 1-2 h at room temperature: horseradish peroxidase conjugated to donkey anti-rabbit (GE Healthcare, NA934V; 1:2,000), horseradish peroxidase conjugated to donkey anti-human (Jackson ImmunoResearch 109-035-149; 1:10,000), horseradish peroxidase conjugated to donkey anti-mouse (GE Healthcare, NA931V; 1:2,000). Blots were washed 3x in PBST and incubated with Amersham ECL detection kit (GE Healthcare). Blots were imaged using chemiluminescence with ImageQuant 400 (GE Healthcare).
Amersham ecl detection kit
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Spain
The Amersham ECL detection kit is a laboratory equipment product designed for the detection and quantification of proteins using the enhanced chemiluminescence (ECL) technique. The kit provides the necessary reagents and components to perform this analysis.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit hrp conjugated secondary antibody, by Merck Group (2 mentions)
Evans blue, by Merck Group (2 mentions)
G box imager, by Syngene (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
A2066, by Merck Group (2 mentions)
Coomassie blue, by Thermo Fisher Scientific (2 mentions)
Pten clone d4.3 xp 9271, by Cell Signaling Technology (2 mentions)
Sigmafast protease inhibitor tablet, by Merck Group (2 mentions)
Visionworks ls software, by Analytik Jena (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal anti α tubulin, by Merck Group (1 mentions)
Imagequant 400, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Calf serum, by Lonza (1 mentions)
Trichrome masson staining reagents, by Merck Group (1 mentions)
Fitc conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti mouse secondary antibody, by Agilent Technologies (1 mentions)
21 protocols using amersham ecl detection kit
1
Immunoblotting of HJURP in Cell Lines
2
Fc-binding minicells display EGFR1 antibody
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Example 3
Binding and display of EGFR1 antibody by minicells expressing the Fc binding portion of Protein A or Protein G. Minicells (1e09) purified from strain VAX13B7 (Protein A-displaying), VAX13C4 (Protein G-displaying), and VAX12B5 (negative control) were incubated with 1 microgram each without (−) or with (+) a mouse monoclonal IgG antibody against human EGFR1 (mAb528) for 1 hour at room temperature to allow antibodies to bind to the minicells. After incubation, minicells were washed three times each with 1 mL of PBS (pH 7.4) to remove any unbound antibody. Minicells (1e08) were then analyzed by Western blot using an HRP-conjugated rabbit anti-mouse polyclonal antibody as the secondary antibody. The Western Blot is shown in
As shown in
3
Analyzing snRNP Levels and Splicing Protein Associations
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Nuclear extracts (200 µg each) were diluted with an equal volume of gradient buffer (G150: 20 mM HEPES pH 7.9, 150 Mm NaCl, 1.5 mM MgCl2 and 0.5 mM DTT) and sedimented on linear 4 ml 10-30% (v/v) glycerol gradients in the G150 buffer. After ultracentrifugation in a Sorvall TH-660 rotor for 14 h at 29,000 rpm (114,000 × g), the gradients were separated into 24 fractions. To analyse the relative levels of snRNPs in the nuclear extracts, proteins in the gradient fractions were digested by Proteinase K in 20 mM HEPES pH 7.9, 150 mMNaCl, 10 mM EDTA, 1% (w/v) SDS for 45 min at 42 °C, the RNAs were extracted by phenol/chloroform/isoamylalcohol and precipitated. The isolated RNAs were separated by denaturing 8% urea PAGE followed by Northern blotting using 5′-end radiolabeled DNA probes against U1, U2, U4, U6 and U5 snRNAs. To analyse the association of selected splicing proteins with the tri-snRNP, proteins were precipitated from gradient fractions and separated on NuPAGE 4−12% Bis–Tris gels (Invitrogen) followed by blotting and immunostaining using antibodies against PRPF8, Brr2, Snu114, PRPF31 (against its C terminus), PRPF4 and SF3b155, and the Amersham ECL detection kit (GE Healthcare). All antibody details are shown in Supplementary Data 6 .
4
Brr2 Variant Sedimentation Analysis
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Yeast cells producing FL or truncated variants of Brr2 were grown in YPD medium at 30°C, and whole-cell extracts were prepared as described (Umen and Guthrie 1995 (link)). Cell extracts were incubated in the absence or presence of 2 mM ATP/MgCl2 for 30 min at 23°C, diluted with an equal volume of the G100 buffer (20 mM HEPES-KOH at pH 7.0, 100 mM KCl, 0.2 mM EDTA), and sedimented on 10%–30% (v/v) glycerol gradients in G100 buffer. The gradients were ultracentrifuged in a Sorvall TST41.14 rotor at 37,000 rpm for 15 h and separated into 27 fractions.
The distributions of Brr2 variants and Snu114 across the gradient fractions were monitored by Western blotting of gradient fractions and immunostaining using antibodies against Brr2 and Snu114 and the Amersham ECL detection kit (GE Healthcare) as previously described (Liu et al. 2015 (link)). To analyze relative levels of snRNPs in cell extracts, proteins in the gradient fractions were digested by proteinase K for 45 min at 37°C in the gradient buffer supplemented with 150 mM NaCl, 10 mM EDTA, and 1% (w/v) SDS. The RNAs were extracted by phenol/chloroform/isoamylalcohol, precipitated by ethanol, and separated by denaturing 8% PAGE followed by Northern blotting using 5′ end radiolabeled DNA probes against U4, U6, and U5 snRNAs (Mozaffari-Jovin et al. 2013 (link)).
The distributions of Brr2 variants and Snu114 across the gradient fractions were monitored by Western blotting of gradient fractions and immunostaining using antibodies against Brr2 and Snu114 and the Amersham ECL detection kit (GE Healthcare) as previously described (Liu et al. 2015 (link)). To analyze relative levels of snRNPs in cell extracts, proteins in the gradient fractions were digested by proteinase K for 45 min at 37°C in the gradient buffer supplemented with 150 mM NaCl, 10 mM EDTA, and 1% (w/v) SDS. The RNAs were extracted by phenol/chloroform/isoamylalcohol, precipitated by ethanol, and separated by denaturing 8% PAGE followed by Northern blotting using 5′ end radiolabeled DNA probes against U4, U6, and U5 snRNAs (Mozaffari-Jovin et al. 2013 (link)).
5
Cardiac Tissue Staining and Analysis
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General cell culture supplies were purchased from BD Biosciences (Spain); calf serum was from BioWhittaker (Verviers, Belgium). Cell culture-grade gelatin, trypsin, antibiotics, hematoxylin-eosin, Trichrome Masson staining reagents, Evans blue, and TTC dyes were from Sigma (Spain). HRP-conjugated anti-mouse secondary antibody and liquid DAB substrate were from Dako (Carpinteria, CA). Anti-MMP-2, MMP-9, CD68, and FITC-conjugated secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-heavy chain cardiac myosin antibody was from Abcam (Abcam, UK). HRP-conjugated anti-rabbit secondary antibody was from Sigma-Aldrich. Amersham ECL detection kit was from GE (GE Healthcare Life Sciences, Spain). Centrifugation concentrators were from Sartorius (Fischer Scientific, Spain).
6
Exosome Protein Characterization Protocol
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Cells were lysed in lysis buffer II (25 mM Tris pH 7.5, 120 mM NaCl, 1% Triton X-100, 1% PSMF, 1 mM NOV, 1 mM Leupeptin) for 1 hour on ice. After centrifugation the protein concentration of the collected supernatants was determined by applying the BCA-assay using bovine serum albumin as standard (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific).
Western blot analysis was accomplished according to standard procedures using 10 μg of cellular protein and a volume of 12 μl exosome lysate corresponding to the exosome amount in 30 ml conditioned medium for SDS polyacrylamide gel electrophoresis. Separated proteins were blotted on nitrocellulose membranes and incubated with primary antibodies directed against CD63 (sc15363, SantaCruz), HSP70 (MA3-007, Affinity Bioreagents), actin (SAB1305567, SIGMA-Aldrich Chemie) and calnexin (sc11397, SantaCruz). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (sc2004 and sc2005, SantaCruz) were used to detect antigen antibody binding via chemoluminescence (Amersham ECL detection kit, GE Healthcare).
Western blot analysis was accomplished according to standard procedures using 10 μg of cellular protein and a volume of 12 μl exosome lysate corresponding to the exosome amount in 30 ml conditioned medium for SDS polyacrylamide gel electrophoresis. Separated proteins were blotted on nitrocellulose membranes and incubated with primary antibodies directed against CD63 (sc15363, SantaCruz), HSP70 (MA3-007, Affinity Bioreagents), actin (SAB1305567, SIGMA-Aldrich Chemie) and calnexin (sc11397, SantaCruz). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies (sc2004 and sc2005, SantaCruz) were used to detect antigen antibody binding via chemoluminescence (Amersham ECL detection kit, GE Healthcare).
7
EGFR1 Antibody Binding to Protein A/G Minicells
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Example 3
Binding and display of EGFR1 antibody by miniciells expressing the Fc binding portion of Protein A or Protein G. Minicells (1e09) purified from strain VAX13B7 (Protein A-displaying), VAX13C4 (Protein G-displaying), and VAX12B5 (negative control) were incubated with 1 microgram each without (−) or with (+) a mouse monoclonal IgG antibody against human EGFR1 (mAb528) for 1 hour at room temperature to allow antibodies to bind to the minicells. After incubation, minicells were washed three times each with 1 mL of PBS (pH 7.4) to remove any unbound antibody. Minicells (1e08) were then analyzed by Western blot using an HRP-conjugated rabbit anti-mouse polyclonal antibody as the secondary antibody. The Western Blot is shown in
As shown in
8
Binding and Display of VEGFR2 Antibody on Minicells
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Example 2
Binding and display of VEGFR2 antibody by miniciells expressing the Fc binding portion of Protein A or Protein G. Minicells (1e09) purified from strain VAX13B7 (Protein A-displaying), VAX13C4 (Protein G-displaying), and VAX12B5 (negative control) were incubated with 1 microgram each without (−) or with (+) a mouse monoclonal IgG antibody against human VEGFR2 for 1 hour at room temperature to allow antibodies to bind to the minicells. After incubation, minicells were washed three times each with 1 mL of PBS (pH 7.4) to remove any unbound antibody. Minicells (1e08) were then analyzed by Western blot using an HRP-conjugated rabbit anti-mouse polyclonal antibody as the secondary antibody. The Western Blot is shown in
9
Evaluating Estrogen Receptor Signaling
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MCF7 cells were serum-starved in phenol red–free RPMI 1640 containing 10% CSS for 5 days. The cells were then seeded onto a six-well plate at a density of 6 × 105 cells/well and treated the following day with the test compounds in the presence of 1 nM E2. After 24 hours, the medium was aspirated, and the cells were washed with ice-cold phosphate-buffered saline. Cells were lysed in 1× radioimmunoprecipitation assay buffer containing one tablet of cOmplete protease inhibitor cocktail (Roche Life Science, Indianapolis, IN, USA). Cell debris was pelleted by centrifugation at 15,000 g for 10 minutes at 4°C. The supernatants were collected and quantified using the BCA assay. In each case, 25 μg of protein was loaded onto 15% (v/v) SDS-PAGE gels, separated and transferred to polyvinylidene fluoride membrane. Membranes were incubated with pS2, cathepsin D and CDC2 antibodies or control α-actin antibody. Bound antibodies were detected using horseradish peroxidase–conjugated secondary antibodies. Chemiluminescence was detected with an Amersham ECL detection kit (GE Healthcare Life Sciences), and bands were visualised using the G:BOX imager (Syngene, Frederick, MD, USA).
10
Spliceosomal Complexes Fractionation
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Yeast cells expressing wt or ts variants of yPrp3 were grown in YPD medium at 30°C. Whole cell extract prepared from each strain was incubated at 37°C (the non-permissive temperature for the Prp3 ts variants) for 30 min, diluted with equal volume of G100 buffer (20 mM HEPES-KOH, pH 7.0, 100 mM KCl, 0.2 mM EDTA) and fractionated by ultracentrifugation on a 12 ml 10–30 % (v/v) glycerol gradient in G100 buffer. Subsequent to ultracentrifugation at 37,000 rpm for 15 hr in a Sorvall TST41.14 rotor, the distribution of spliceosomal U4, U5 and U6 snRNPs across the gradient fractions was monitored by Northern blotting and quantified as described previously (Mozaffari-Jovin et al., 2013 (link)). The association of yPrp3 with tri-snRNP was analyzed by Western blotting of gradient fractions and immunostaining using antibodies against yPrp3 and ySnu114 and the Amersham ECL detection kit (GE Healthcare).
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