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Enhanced chemiluminescence

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Enhanced chemiluminescence is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It relies on the emission of light produced by a chemical reaction, which is then measured and analyzed to provide information about the target analyte.

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Lab products found in correlation

Nitrocellulose membrane, by Bio-Rad (8 mentions) Pvdf membrane, by Merck Group (8 mentions) Horseradish peroxidase secondary antibody, by Jackson ImmunoResearch (6 mentions) Protease inhibitor cocktail, by Merck Group (6 mentions) Gapdh, by Santa Cruz Biotechnology (4 mentions) β actin, by Santa Cruz Biotechnology (4 mentions) β actin, by Merck Group (4 mentions) Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (4 mentions) Anti cleaved caspase 3, by Cell Signaling Technology (3 mentions) Non fat dried milk, by Bio-Rad (3 mentions) Anti gapdh, by Cell Signaling Technology (3 mentions) Protease inhibitor cocktail, by Roche (3 mentions) E cadherin, by Cell Signaling Technology (3 mentions) Goat anti mouse or anti rabbit hrp conjugated secondary antibodies, by Cell Signaling Technology (3 mentions) Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (3 mentions) Immobilon p pvdf membrane, by Merck Group (3 mentions) Nitrocellulose membrane, by Cytiva (3 mentions) Trizol reagent, by Thermo Fisher Scientific (2 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Dc protein assay reagent, by Bio-Rad (2 mentions)

148 protocols using enhanced chemiluminescence

1

Western Blot Protein Detection

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As described in study (20) , the whole protein were collected in denaturing SDS sample buffer and analyzed by western blotting, and transferred to a 0.22-µm nitrocellulose transfer membrane. The membrane was blocked with 5% (w/v) milk in PBS/0.05% (v/v) Tween-20 and incubated with the indicated antibody overnight at 4°C followed by incubation with a horseradish peroxidase secondary antibody (Jackson ImmunoResearch)
for 1 h at room temperature. Proteins were detected using an enhanced chemiluminescence (Perkin Elmer).
Xie Y., Huang S., Liu T., Sun H., Ren H., & Xie C. (2020). All-Trans Retinoic Acid, A Derivative of Vitamin A, Improved Intestinal Epithelial Barrier Function Through PFKP.
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2

Detecting Protein Expression Levels

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Cell lysates were prepared and processed as described [16 (link)]. Antibodies were used at the following dilutions: β-actin (MAB1501, 1:1,000; Chemicon), KDM6A (ab36938, 3μg/mL; Abcam), p21CIP1 (ab109520, 1:750 and ab109199, 1:1,000; Abcam), FLAG (F-3165, 1:1,000; Sigma) and HRP-conjugated secondary anti-rabbit (1:10,000; Amersham) and anti-mouse (1:10,000; Amersham). Antigen/antibody complexes were visualized by enhanced chemiluminescence (PerkinElmer Life Sciences) and electronically acquired with a Kodak 4000R Image Station (Kodak) equipped with Carestream Molecular Imaging Software.
Soto D.R., Barton C., Munger K, & McLaughlin-Drubin M.E. (2017). KDM6A addiction of cervical carcinoma cell lines is triggered by E7 and mediated by p21CIP1 suppression of replication stress. PLoS Pathogens, 13(10), e1006661.
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3

Verifying Protein Expression and mRNA Levels

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The protein expression of selected candidates was verified by Western blotting according to standard procedures using specific antibodies (see supplemental Table S1) that were detected with HRP-conjugated secondary antibodies and enhanced chemiluminescence (PerkinElmer Life Sciences; Beaconsfield, UK). Real time qRT-PCR was used to determine IRF9 mRNA expression in HMLECs following IFNγ and IFNγ plus EGF treatment. Total RNA was extracted from cells using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. 2.5 μg of total RNA was used for reverse transcription using random hexamer primers (Applied Biosystems; Warrington, UK) and Superscript II reverse transcriptase (Invitrogen) following the manufacturer's protocol, and real time qRT-PCR was carried out using a TaqMan Gene Expression assay specific for IRF9 (Applied Biosystems). Reactions were run on an ABI Prism 7700 sequence detection system using standard cycling conditions. Ct values were determined, and the standard curve method using the HB4a control sample as calibrator and endogenous 18S mRNA as control was used to calculate relative mRNA expression.
Worthington J., Spain G, & Timms J.F. (2017). Effects of ErbB2 Overexpression on the Proteome and ErbB Ligand-specific Phosphosignaling in Mammary Luminal Epithelial Cells. Molecular & Cellular Proteomics : MCP, 16(4), 608-621.
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4

Western Blot Analysis of Protein Phosphorylation

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A single cell suspension was obtained from spleens and red cells lysed with 0.155 M NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA (pH 7.5). Cell lysates were prepared and equal amounts of protein loaded in each lane of 7.5 or 15% SDS-PAGE gels and transferred onto nitrocellulose membranes as previously described [22 (link)]. Phosphorylated and total proteins were detected sequentially on the same membrane using specific primary antibodies, appropriate secondary antibodies conjugated to HRP and enhanced chemiluminescence (Perkin Elmer, Boston, MA). Bands were quantitated by densitometry (Molecular Dynamics) using ImageQuant software.
Wong J., Welschinger R., Hewson J., Bradstock K.F, & Bendall L.J. (2014). Efficacy of dual PI-3K and mTOR inhibitors in vitro and in vivo in acute lymphoblastic leukemia. Oncotarget, 5(21), 10460-10472.
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5

Western Blot Analysis of Cellular Proteins

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Cell lysates were prepared by using RIPA buffer containing 1 mM PMSF, final concentration. Cell lysates (50 μg protein) were loaded into each well and separated by electrophoresis on a 12% SDS-PAGE gel (Bio-Rad). Gel separated samples were transferred to a 0.2 μm pore-sized membrane (Bio-Rad) by using a semi-dry transfer (40 mA, per membrane/gel), for 45 min. The membrane was dried in methanol for 15 min. and blocked in 5% dry milk dissolved in TBST (Tris Buffer Saline + Tween) for 2 hrs at room temperature, followed by overnight incubation with antibodies (Table 1) at 4°C. The membrane was washed three times in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The blot was developed by using enhanced chemiluminescence, from Perkin-Elmer (Boston, MA, USA). In the western blot experiments analysing ubiquitinylated proteins and glutathionylated proteins, 5 and 10 μg of protein were loaded per sample lane, respectively. In these experiments, the membrane was blocked with 1% BSA.
Wallenberg M., Misra S., Wasik A.M., Marzano C., Björnstedt M., Gandin V, & Fernandes A.P. (2014). Selenium induces a multi-targeted cell death process in addition to ROS formation. Journal of Cellular and Molecular Medicine, 18(4), 671-684.
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6

Detecting Cilostazol-Induced CXCR4 and SDF-1α Expression

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To detect cilostazol-stimulated expression of CXCR4 and SDF-1α proteins, EPCs were preincubated with various concentrations of cilostazol in M199 supplemented with 0.5% serum for 24 hours before protein harvesting. Proteins were separated by SDS-PAGE (10% polyacrylamide gel) and transferred to PVDF membranes, which were preincubated with antibodies against CXCR4, SDF-1α, and actin. Antibody binding was detected with HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and enhanced chemiluminescence (PerkinElmer, Shelton, CT, USA), followed by exposure to X-ray film.
Tseng S.Y., Chao T.H., Li Y.H, & Cho C.L. (2016). Cilostazol Improves Proangiogenesis Functions in Human Early Endothelial Progenitor Cells through the Stromal Cell-Derived Factor System and Hybrid Therapy Provides a Synergistic Effect In Vivo. BioMed Research International, 2016, 3639868.
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7

Quantifying Protein Expression in Muscle Tissue

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Homogenates of muscle tissues were prepared and proteins (40 μg per lane) were separated by SDS-PAGE (10% polyacrylamide gel) and transferred to PVDF membranes. Membranes were incubated with antibodies against phospho-eNOS, eNOS, phospho-Akt, Akt, SDF-1α, and CXCR4. Antibody binding was detected with HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and enhanced chemiluminescence (PerkinElmer, Shelton, CT, USA), followed by exposure to X-ray film.
Tseng S.Y., Chao T.H., Li Y.H, & Cho C.L. (2016). Cilostazol Improves Proangiogenesis Functions in Human Early Endothelial Progenitor Cells through the Stromal Cell-Derived Factor System and Hybrid Therapy Provides a Synergistic Effect In Vivo. BioMed Research International, 2016, 3639868.
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8

Protein Carbonylation Quantification

Cited 1 time
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IPG strips were incubated in 2 N HCl with 10 mM DNPH at 25°C for 15 min after IEF; strips were then washed with 2 M Tris-Base/30% glycerol for 15 min [24 (link)]. The IPG strips were used for molecular weight-dependent separation of proteins by SDS-PAGE and transferred the protein blotting to a membrane which was incubated overnight at 4°C with the anti-DNP antibody in TBST containing 5% milk. The blots were washed and incubated goat anti-rabbit IgG HRP conjugate for 2 hrs. Enhanced chemiluminescence (PerkinElmer, CA, USA) was used for detection.
Hung Y.C., Wang P.W., Lin T.Y., Yang P.M., You J.S, & Pan T.L. (2020). Functional Redox Proteomics Reveal That Salvia miltiorrhiza Aqueous Extract Alleviates Adriamycin-Induced Cardiomyopathy via Inhibiting ROS-Dependent Apoptosis. Oxidative Medicine and Cellular Longevity, 2020, 5136934.
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9

Western Blot Protein Analysis Protocol

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Samples proteins were isolated by cell lysis buffer (Cell Signaling, MA, USA) and measured using the Bradford Protein Assay Kit (AMRESCO, OH, USA). Total proteins were separated with 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane (PALL). Next, the blots were incubated with specific primary antibody overnight at 4°C after blocking and further incubated with a peroxidase-labeled anti-mice or -rabbit IgG for 2 h; blots were then washed and incubated goat anti-rabbit and anti-mouse IgG (Chemicon) HRP conjugate for 2 hrs. Enhanced chemiluminescence (PerkinElmer, USA) was used for signal detection. The level of expression of β–actin was used as a gel loading control [27 (link)].
Hung Y.C., Wang P.W., Lin T.Y., Yang P.M., You J.S, & Pan T.L. (2020). Functional Redox Proteomics Reveal That Salvia miltiorrhiza Aqueous Extract Alleviates Adriamycin-Induced Cardiomyopathy via Inhibiting ROS-Dependent Apoptosis. Oxidative Medicine and Cellular Longevity, 2020, 5136934.
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10

Protein Expression Analysis by Western Blot

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Total cell lysate was prepared with radioimmunoprecipitation assay buffer containing Protease Inhibitor Cocktail. Protein concentrations were measured by DC protein assay reagent (Bio-Rad, CA, USA) and extracts resolved by SDS/PAGE on 8% gels. Membranes were blocked for 2 h at room temperature in tris buffered saline-tween-20 containing 5% nonfat dried milk (Bio-Rad), washed, and then incubated with primary antibodies (anti-EGFR from Santa Cruz, CA, USA; anti-pErbB2, anti-Met, anti-Akt, anti-pAkt, anti-GAPDH, anti-p27/kip1, anti-cleaved-caspase 3, anti-bax, and anti-bcl-2 from Cell Signaling Technology, Boston, USA) at 4°C overnight. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Boston, USA). The signal was detected using enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA).
Jian F.F., Li Y.F., Chen Y.F., Jiang H., Chen X., Zheng L.L., Zhao Y., Wang W.Q., Ning G., Bian L.G, & Sun Q.F. (2016). Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Adrenocorticotropic Hormone Production and Tumorous Corticotroph Cell Growth in AtT20 Cells. Chinese Medical Journal, 129(17), 2102-2108.
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