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Amersham ecl western blot reagent

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The Amersham ECL Western blot reagent is a chemiluminescent detection system used in Western blot analysis to visualize and quantify specific proteins in a sample. The reagent produces a luminescent signal when it reacts with the enzyme-labeled secondary antibody, allowing for the detection of target proteins.

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Lab products found in correlation

Ab8224, by Abcam (3 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Sc 27046, by Santa Cruz Biotechnology (1 mentions) Phosstop phosphatase inhibitor cocktail, by Merck Group (1 mentions) Pa5 17869, by Thermo Fisher Scientific (1 mentions) Ab290, by Abcam (1 mentions)

Close spelling variants of this product

Amersham ECL Prime Western blot detection reagent Amersham ECL Select Western Blot Detection Reagent Amersham ECL Western blot detection reagent Amersham ECL reagent AmershamTM ECLTM Amersham ECL ECL reagent

5 protocols using amersham ecl western blot reagent

1

Apoptosis Markers in X. laevis with 3q29 Knockdown

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Two replicate western blot experiments were performed to test for apoptosis markers in X. laevis with 3q29 gene knockdown (S12 Fig). Embryos at stages 20–22 were lysed in buffer (50mM Tris pH 7.5, 1% NP40, 150mM NaCl, 1mM PMSF, 0.5 mM EDTA) supplemented with cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, Basel, Switzerland). Blotting was carried out using rabbit polyclonal antibody to cleaved caspase-3 (1:500, 9661S, Cell Signaling Technology, Danvers, MA, USA), with mouse anti-beta actin (1:2500, AB8224, Abcam, Cambridge, UK) as a loading control on a Mini-PROTEAN TGX precast 4–15% gradient gel (Bio-Rad, Hercules, CA, USA). Chemiluminescence detection was performed using Amersham ECL western blot reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Band intensities were quantified by densitometry in ImageJ and normalized to the control mean relative to beta-actin. Due to the low number of replicates, we did not perform any statistical tests on data derived from these experiments.
Singh M.D., Jensen M., Lasser M., Huber E., Yusuff T., Pizzo L., Lifschutz B., Desai I., Kubina A., Yennawar S., Kim S., Iyer J., Rincon-Limas D.E., Lowery L.A, & Girirajan S. (2020). NCBP2 modulates neurodevelopmental defects of the 3q29 deletion in Drosophila and Xenopus laevis models. PLoS Genetics, 16(2), e1008590.
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2

TACC3 and XMAP215 Immunoblotting

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Embryos were lysed in buffer (50 mM Tris, pH 7.5, 5% glycerol, 0.2% Igepal, 1.5 mM MgCl2, 125 mM NaCl, 25 mM NaF, 1 mM Na3VO4, 1 mM dithiothreitol, supplemented with Complete Protease Inhibitor cocktail with or without EDTA [Roche, Indianapolis, IN]). For collection of neural/axonal lysates, neural tube explants were grown on coverslips overnight. Medium was then removed and replaced with lysis buffer (as described), and cultures were scraped to obtain the neural tissue. Blotting was carried out using goat anti–X. laevis TACC3 (1:500; sc-27046; Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-XMAP215 (1:2500; a gift from the Hyman lab). Mouse anti–β-actin (1:2500; ab8224; Abcam) was used as a loading control. For TACC3 detection, 1% immunoglobulin G (IgG)-free bovine serum albumin (BSA) in phosphate-buffered saline/Tween-20 (PBST) was used to block nitrocellulose membrane. For other antibodies, 2% nonfat milk was used. Detection was done by chemiluminescence using Amersham ECL Western Blot reagent (GE Healthcare, Pittsburgh, PA). The bands were quantified by densitometry using Photoshop (Adobe, San Jose, CA), and the data and graphs were compiled in Excel (Microsoft, Redmond, WA).
Nwagbara B.U., Faris A.E., Bearce E.A., Erdogan B., Ebbert P.T., Evans M.F., Rutherford E.L., Enzenbacher T.B, & Lowery L.A. (2014). TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types. Molecular Biology of the Cell, 25(21), 3350-3362.
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3

Quantifying Phospho-Histone H3 in Embryos

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Embryos at stage 20 to 22 were lysed in buffer (50mM Tris pH 7.5, 1% NP40, 150mM NaCl, 1mM PMSF, 0.5 mM EDTA), supplemented with cOmplete™ Mini EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) and PhosSTOP Phosphatase Inhibitor Cocktail (Sigma-Aldrich). Blotting was carried out using rabbit polyclonal antibody to Phospho-Histone H3 (Ser10) (1:500, PA5-17869, Invitrogen), with mouse anti-beta actin (1:2500, ab8224, Abcam, Cambridge, MA, USA) as a loading control. Bands were detected by chemiluminescence using Amersham ECL Western blot reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA). Band intensities were quantified by densitometry in ImageJ and normalized to the control mean relative to β-actin.
Pizzo L., Lasser M., Yusuff T., Jensen M., Ingraham P., Huber E., Singh M.D., Monahan C., Iyer J., Desai I., Karthikeyan S., Gould D.J., Yennawar S., Weiner A.T., Pounraja V.K., Krishnan A., Rolls M.M., Lowery L.A, & Girirajan S. (2021). Functional assessment of the “two-hit” model for neurodevelopmental defects in Drosophila and X. laevis. PLoS Genetics, 17(4), e1009112.
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4

Western Blot of GFP in Embryos

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Embryos were lysed in buffer (50 mM Tris pH 7.5, 5% glycerol, 0.2% IGEPAL, 1.5 mM MgCl2, 125 mM NaCl, 25 mM NaF, 1 mM Na3V04, 1 mM DTT), supplemented with complete protease inhibitor cocktail with or without EDTA (Roche, Indianapolis, IN). Blotting was carried out using rabbit anti-GFP [1:2500, ab290, Abcam (Cambridge, MA)]. For blocking membrane, 2% nonfat milk was used. Detection was done by chemiluminescence using Amersham ECL Western blot reagent (GE Healthcare, Pittsburgh, PA).
Lucaj C.M., Evans M.F., Nwagbara B.U., Ebbert P.T., Baker C.C., Volk J.G., Francl A.F., Ruvolo S.P, & Lowery L.A. (2015). Xenopus TACC1 is a microtubule plus‐end tracking protein that can regulate microtubule dynamics during embryonic development. Cytoskeleton (Hoboken, N.j.), 72(5), 225-234.
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5

Tacc3 Depletion and Rescue in Cells

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Capped mRNAs were transcribed and purified as previously described (Lowery et al., 2013; Nwagbara et al., 2014) . Morpholino antisense oligonucleotides were used to target Tacc3 (5-AGTTGTAGGCTCATTCTAAACAGGA3), used and validated previously (Nwagbara, Faris et al. 2014) . Morpholino KD conditions were always compared to cells from embryos injected with standard control morpholino (5-CCTCTTACCTCAGTTACAATTTATA-3) (Gene Tools, LLC). Tacc3 depletion was validated by Western Blot, as previously described (Nwagbara, Faris et al. 2014) . Band detection was by chemiluminescence using Amersham ECL Western blot reagent (GE Healthcare BioSciences), and relative depletion was quantified by band densitometry using ImageJ. Rescue experiments were performed with exogenous mRNAs co-injected with their corresponding MOs. Plasmid for TACC3, cloned into pET30a, was a kind gift from the Richter lab (University of Massachusetts Medical School, Worcester, MA), which was subcloned into pCS2. As a start-site morpholino was utilized to block Tacc3 translation, a morpholino-resistant exogenous mRNA was generated by creating conserved mutations in the first 7 codons.
Bearce E.A., Pratt B.C., Rutherford E.L., Carandang L., & Lowery L.A. (2021). Tacc3 modulates microtubule network dynamicity and focal adhesion remodeling to affect cranial neural crest cell migration in Xenopus laevis.
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