RPMI-1640 culture medium and FBS were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). L-glutamine, salt pyruvate, non-essential amino acids, streptomycin, phosphate-buffered saline (PBS) and nuclear fluorochrome Hoechst 33342 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against RhoA (monoclonal mouse; #sc-418; dilution, 1:200) and fibrillarin (mouse monoclonal; #sc-166000; dilution, 1:100) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), whereas monoclonal mouse antibody against B23 (#B0556; dilution, 1:100) was purchased from Sigma-Aldrich. Actinomycin D was additionally obtained from Sigma-Aldrich. Mouse monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH; #KC-5G4; dilution, 1:5,000) was obtained from KangCheng Bio-tech Inc. (Shanghai, China). Goat anti-mouse immunoglobulin (Ig)G fluorescein isothiocyanate-conjugated (FITC; #115-097-003; dilution, 1:1,000) and goat anti-mouse IgG horseradish peroxidase (HRP)-conjugated (#115-035-209; dilution, 1:5,000) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from GE Healthcare Life Sciences (Chalfont, UK).
Enhanced chemiluminescence ecl reagent
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Enhanced chemiluminescence (ECL) reagents are a type of laboratory equipment used for the detection and quantification of proteins in Western blot analysis. ECL reagents produce a luminescent signal when reacted with the enzyme horseradish peroxidase, which is commonly used to label target proteins. This luminescent signal can then be detected and measured using specialized imaging equipment.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
X ray film, by GE Healthcare (2 mentions)
Fibrillarin, by Santa Cruz Biotechnology (1 mentions)
Non essential amino acids (neaa), by Merck Group (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Fitc conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Fitc conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Hrp conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Streptavidin fitc, by Merck Group (1 mentions)
Pflag ctc vector, by Merck Group (1 mentions)
Hrp conjugated rabbit anti mouse igg, by Jackson ImmunoResearch (1 mentions)
3h oleic acid, by PerkinElmer (1 mentions)
Immunoblotting reagents, by Bio-Rad (1 mentions)
Dovitinib, by Selleck Chemicals (1 mentions)
15 protocols using enhanced chemiluminescence ecl reagent
1
Immunofluorescence Analysis of RhoA and Nucleolar Proteins
2
Ginkgol C17:1 Biochemical Assay
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Trypsin-EDTA solution was purchased from Gibco; Thermo Fisher Scientific, Inc. The horseradish peroxidase (HP)-conjugated secondary antibodies, goat anti-mouse polyclonal IgG (cat. no. A0216; dilution, 1:10,000) and goat anti-rabbit polyclonal IgG (cat. no. A0208; dilution, 1:10,000) were purchased from Beyotime Institute of Biotechnology (Haimen, China). The enhanced chemiluminescence (ECL) reagents were bought from GE Healthcare Life Sciences (Chalfont, UK). MTT was purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). Mouse monoclonal immunoglobulin G (IgG) anti-as homolog gene family, member A (RhoA; cat. no. sc-418; dilution, 1:1,000) and β-actin antibody (cat. no. sc-47778; dilution, 1:1,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyclonal rat anti-rabbit matrix metalloproteinase (MMP)-7 antibody (cat. no. S247; dilution, 1:1,000) was purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA). Rabbit polyclonal antibodies against protein kinase B (Akt; cat. no. IM001-0359; dilution, 1:1,000) and phosphorylated (p)-Akt (cat. no. IM001-0270; dilution, 1:1,000) were purchased from ExCel Biology Co., Ltd. (Shanghai, China). Ginkgol C17:1 (high performance liquid chromatography (HPLC) purity of >96.5%) was donated by the School of Food and Biological Engineering, Jiangsu University (7 ).
3
Investigating Cell Adhesion Molecules
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MOPC-21 (IgG1), pFLAG-CTC vector, and Streptavidin-FITC were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-CD44 (clone 156-3C11) and anti-α2 integrin subunit (clone P1E6, IgG1) antibodies were purchased from Millipore (Billerica, MA, USA). FITC-conjugated goat anti-mouse IgG, FITC-conjugated goat anti-rabbit IgG, HRP-conjugated rabbit anti-mouse IgG, HRP-conjugated rat anti-rabbit IgG, and HRP-conjugated Streptavidin were purchased from Jackson Immunoresearch (West Glove, PA, USA). Anti-CD44v10 antibody (AB2082) was purchased from Calbiochem (San Diego, CA, USA). Anti-FLAG (M2) antibody was purchased from Stratagene (Santa Clara, CA, USA). Type I Collagen was purchased from Inamed Biomatenak (Freemont, CA, USA). Enhanced chemiluminescence (ECL) reagents were purchased from GE Healthcare (Piscataway, NJ, USA). Other chemicals were purchased from Sigma-Aldrich unless otherwise mentioned.
4
Oleic Acid Uptake in Rat Cells
5
Western Blot Analysis of Protein Signaling
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Lysates were collected in radioimmunoprecipitation assay buffer [RIPA; 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% TritionX-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, and 10 μg/mL aprotinin]. 25 μg or 30 μg of total protein was separated by 10% or 12.5% SDS-PAGE followed by transfer to Immobilon-P membrane. Immunoblotting reagents were from the following sources: antibodies against p-FGFR (Tyr653/654), FGFR1 (D8E4), FGFR4 (D3B12), p-VEGFR (19A10), VEGFR2 (55B11), p-AKT (D9E), AKT (9272), p-MAPK (D13.14.4E), p44/42 MAPK (Erk1/2), OCT4 (2750), CD133 (D2V8Q), Androgen Receptor (D6F11), and β-actin (4967) antibodies were from Cell Signaling Technology; FGFR2 (C-8), FGFR3 (B-9), and STAT5 (C-17) were from Santa Cruz Biotechnology; ALDH7A1 (CAT:ABO11656) was from Abgent; HRP anti-mouse, HRP anti-rabbit, and Enhanced Chemiluminescence (ECL) reagents were from GE Healthcare. Other reagents included: Dovitinib, BGJ398 and PD166866 were from Selleckchem.
6
Western Blotting Protocol for Protein Analysis
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Western blotting was performed either with the Simple Western assay by Simon (ProteinSimple, Santa Clara, CA, USA) according to manufacturer instruction [54 (link)] or as previously described [13 ]. Antibodies used were as follows: total RNAPII, phosphorylated RNAPII serine-2 (p-RNAPIIS2) and serine-5 (p-RNAPIIS5) (Covance, NJ, USA), 4E-BP1, p-4E-BP1 (pT70), β-Actin, caspase-3, caspase-7, CDK9, eIF4E, p-eIF4E (S209), eIF4G, p-ErkT202/Y204, p-p38T180/Y182, p38 MAPK, rpS6, Mcl-1, Mnk1, PARP, cleaved PARP (Cell Signalling Technology, Danvers, MA, USA), Erk (ProteinSimple), MDM-2 (Becton Dickenson, Franklin Lakes, NJ, USA), Bcl-2, cyclin D1, p-S6S240/S244, p53 (Dako, Glostrap, Denmark). Both anti-mouse and anti-rabbit immunoglobulin G (IgG) horseradish peroxidase conjugated antibodies were obtained from Dako. Enhanced Chemiluminescence (ECL) reagents were obtained from GE Life Sciences.
7
Western Blotting of PI4KIIα in Cells
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All cell culture materials, enhanced chemiluminescence (ECL) reagents and X-ray film were purchased from GE Healthcare Life Sciences, UK. Polyvinylidene difluoride (PVDF) membrane was bought from Merck Millipore UK. Horseradish peroxidase (HRP)-linked secondary antibodies were purchased from Cell Signalling Technology UK. The antibody to PI4KIIα was previously described by us (Minogue et al., 2010 (link)). HRP-linked cholera toxin B subunit was purchased from Sigma-Aldrich UK. Sucrose was obtained from VWR International Ltd UK. Complete protease inhibitor tablets were purchased from Roche Ltd UK. All other reagents were from Sigma-Aldrich UK.
8
Detailed Biochemical Protocol for NR Synthesis
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Unless otherwise specified, all chemicals and reagents were of analytical grade and were purchased from Sigma and Amresco. Cell culture reagents were from Gibco, Greiner Bio-One, and Orange Scientific. HPLC-grade methanol and acetonitrile were obtained from Merck. The ultrapure water was obtained from a Milli-Q Synthesis purification system (Millipore). NR was synthesized as reported previously (52 (link)). DNA-modifying and restriction enzymes were purchased from Thermo Fisher Scientific. The following antibodies were used: rabbit anti-PNP (Sigma), mouse anti-FLAG (Sigma), mouse anti-β-tubulin (Sigma), rabbit anti-α-tubulin (Abcam), mouse anti-α-acetylated (K40) tubulin (Sigma), HRP-conjugated rabbit anti-mouse (Sigma), and HRP-conjugated goat anti-rabbit (Sigma). Enhanced chemiluminescence (ECL) reagents were from GE Healthcare.
9
Western Blot Quantification of Protein Expression
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The relative protein expression levels of the genes of interest were determined using western blotting. An aliquot of 50 µg of total cellular proteins was separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then blotted onto a piece of BioTrace™ polyvinylidene difluoride (PVDF) membrane (Pall Corporation, Pensacola, FL, USA). Non-specific bindings were blocked with 5% skimmed milk in a Tris-buffered saline Tween-20 (TBST) buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween-20, pH 7.4). The membrane was subjected to specific primary antibodies against target proteins, as listed in Table II, and was incubated for 16 h at 4̊C (22, 50) . β-actin was used as the internal control. The membrane was then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature, the protein band was visualized on X-ray film (Fujifilm, Tokyo, Japan) using enhanced chemiluminescence (ECL) reagents (GE Healthcare, Chalfont, UK). The band density was scanned by a densitometer to calculate the relative protein expression levels. Each experiment was repeated using three batches of cultured cells (n=3).
10
Immunoblotting of signaling proteins
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2×107 cells were lysed using 350–400 µL lysis buffer (Pierce, Rockford, IL) supplemented with protease and phosphatase inhibitors (Sigma, St. Louis, MO), and lysates were cleared by centrifugation at 13,000 rpm for 30 min at 4°C. Equal amounts of total protein lysates (15 µg) were resolved by SDS-PAGE, transferred onto PVDF membranes and probed with antibodies. c-Cbl (C-15) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). pS621c-Raf antibody was from Pierce Thermo Scientific (Lafayette, CO). Lyn, Fgr, pY416-SFK, AhR, Vav1, Slp76, p47phox, c-Raf, pS259c-Raf, pS289/296/301c-Raf, VDR, RARα, GAPDH, horseradish peroxidase anti-mouse and horseradish peroxidase anti-rabbit were from Cell Signaling (Danvers, MA, USA). Enhanced chemiluminescence ECL reagent (GE Healthcare, Pittsburg, PA) was used for detection.
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