Chemiluminescence kit

Manufactured by PerkinElmer

Sourced in United States

The Chemiluminescence kit is a laboratory equipment designed to detect and quantify the presence of specific analytes in a sample through the process of chemiluminescence. The kit includes the necessary reagents and components to perform this analysis.

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Lab products found in correlation

Semidry transfer system, by Thermo Fisher Scientific (3 mentions) Nitrocellulose or pvdf membranes, by Bio-Rad (3 mentions) Bca protein assay kit, by Thermo Fisher Scientific (3 mentions) Hrp conjugated secondary antibody, by MultiSciences Biotech (2 mentions) Protease inhibitor cocktail, by Merck Group (2 mentions) Ripa buffer, by Beyotime (2 mentions) β actin, by Merck Group (2 mentions) Flag tag (flag), by Merck Group (1 mentions) Polyvinylidene difluoride membrane, by PerkinElmer (1 mentions) Tubulin, by Merck Group (1 mentions) P src, by Cell Signaling Technology (1 mentions) Itgav, by Cell Signaling Technology (1 mentions) Ab68153, by Abcam (1 mentions) Ab72608, by Abcam (1 mentions) A5316, by Merck Group (1 mentions) Ab76315, by Abcam (1 mentions) β actin, by Abcam (1 mentions) P stat3, by Abcam (1 mentions) Un scan it gel ver 6, by Silk Scientific (1 mentions) Anti phospho gsk3, by Cell Signaling Technology (1 mentions)

17 protocols using chemiluminescence kit

Characterization of BEAF and FLAG Proteins via 1D/2D Western and Southwestern Blotting

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NE was analyzed by 1D/2D SDS-PAGE, transferred to PVDF membrane and probed with antibodies to BEAF (DSHB, dilution 1:1000) or FLAG (Sigma, dilution 1:5000). The blots were developed using chemiluminescence kit from Perkin Elmer as per the manufacturer's protocol. 2D gel electrophoresis was performed using the Protean II electrophoresis setup (Bio-Rad). NE (100 µg) was resolved on the Ready Strips IPG strip from Bio-Rad (3–10 NL). The 2D run was performed on a 12% SDS-polyacrylamide slab gel. 2D gels were also processed for western blotting as described above.
For southwestern blotting, proteins resolved on 2D-gel were transferred to PVDF membrane. Proteins on membrane were denatured with 8M GnHCl for 10 min and slowly re-natured by diluting the GnHCl in a stepwise manner. The membrane was blocked in 5% BSA and hybridized to ³²P-labeled scs' probe DNA overnight. The blot was washed stringently and exposed to phosphor imaging screen to obtain the image.

De D., Kallappagoudar S., Lim J.M., Pathak R.U, & Mishra R.K. (2017). O-GlcNAcylation of boundary element associated factor (BEAF 32) in Drosophila melanogaster correlates with active histone marks at the promoters of its target genes. Nucleus, 9(1), 65-86.

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Protein Expression and Detection

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Vero cells grown on 35-mm dishes were transfected as described above with 1 µg of different plasmids. At 24 h pt, cells were scraped on ice into NP-40 lysis buffer (10 mM EGTA, 2.5 mM MgCl2, 1% NP-40, 20 mM HEPES pH 7.4) and sonicated. Equal volumes of each sample mixed with Laemmli sample buffer were boiled, separated by SDS-PAGE 12%, and transferred onto a nitrocellulose membrane. The membrane was blocked, and proteins were detected by incubation with the selected primary antibody and the corresponding horseradish peroxidase-coupled secondary antibody that was developed using a chemiluminescence kit (Perkin-Elmer).

González-Magaldi M., Martín-Acebes M.A., Kremer L, & Sobrino F. (2014). Membrane Topology and Cellular Dynamics of Foot-and-Mouth Disease Virus 3A Protein. PLoS ONE, 9(10), e106685.

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Protein Expression Analysis by Western Blot

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After EGFR TKI or RNA interference treatments, PC9 or H1975 cells were lysed in the lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris, pH 8.0). The cell lysates were analyzed by means of a BCA protein assay (Pierce, Rockford, IL, USA), resolved by SDS-PAGE, and transferred onto a polyvinylidene difluoride membrane (PerkinElmer Inc., Waltham, MA, USA). The membrane was blocked in a blocking buffer (3% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20 [TBST]) for 1 hour at room temperature. Primary antibodies against β-actin (EMD Millipore, Billerica, MA, USA), tubulin (Sigma-Aldrich, St Louis, MO, USA), p-Src, Src, ITGAV, and ITGB1 (all from Cell Signaling, Danvers, MA), fibronectin (FN1), and p-FAK and FAK (all from GeneTex, Irvine, CA, USA) were diluted in blocking buffer and added to the membrane, which was gently shaken overnight at 4°C. After washing three times with TBST, the membrane was immersed in horseradish peroxidase-conjugated secondary antibody diluted in blocking buffer for 1 hour at room temperature and then washed with TBST. Washed membranes were visualized with a chemiluminescence kit (PerkinElmer Inc.).

Wang H.Y., Hsu M.K., Wang K.H., Tseng C.P., Chen F.C, & Hsu J.T. (2016). Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses. OncoTargets and therapy, 9, 2961-2973.

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Quantification of Protein Biomarkers

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Ice-cold radio-immuno-precipitation assay (RIPA) buffer was applied to extract the proteins from tissue homogenates, either from the urinary bladder or heart. Western blotting analysis was subsequently performed using our previous method [31 (link)]. The target antigens from the protein extracts were characterized using the primary antibodies specific for p-STAT3 (1:1000, phosphor-Y705, ab76315, Abcam), STAT3 (1:1000, ab68153, Abcam), TGR5 (1:1000, ab72608, Abcam), or β-actin (1:5000, A5316, Sigma-Aldrich, St. Louis, MO, USA). The bound primary antibodies were then hybridized to horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgGs (Calbiochem, San Diego, CA, USA), and the immunoreactive bands were developed with a chemiluminescence kit (Perkin Elmer, Waltham, MA, USA). Based on the optical densities of each band, p-STAT3 (88 kDa), STAT3 (88 kDa), TGR5 (35 kDa), or β-actin (43 kDa) was quantified using our previous method [31 (link)].

Hsu C.C., Cheng K.C., Li Y., Hsu P.H., Cheng J.T, & Niu H.S. (2021). TGR5 Expression Is Associated with Changes in the Heart and Urinary Bladder of Rats with Metabolic Syndrome. Life, 11(7), 695.

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Western Blot Analysis of BHK-21 Cells

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BHK-21 cells incubated with peptides and infected were collected and processed as in [7 (link)]. Briefly, equal volumes of each sample were loaded on a SDS-PAGE 12%, transferred onto a nitrocellulose membrane and the proteins detected by incubation with the selected primary antibody and the corresponding horseradish peroxidase-coupled secondary antibody that was developed using a chemiluminescence kit (Perkin-Elmer).

González-Magaldi M., Vázquez-Calvo Á., de la Torre B.G., Valle J., Andreu D, & Sobrino F. (2015). Peptides Interfering 3A Protein Dimerization Decrease FMDV Multiplication. PLoS ONE, 10(10), e0141415.

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Western Blot Analysis of Cardiac Proteins

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We used ice-cold radio-immuno-precipitation assay (RIPA) buffer to extract the proteins from rat heart homogenates or cell lysates. Western blot analysis was subsequently performed according to our previous method [59 (link)]. The target antigens from the protein extracts were detected using primary antibodies specific for PPARδ (Thermo-Fisher Sci., Rockford, IL, USA), p-STAT3, STAT3, CCN2, fibronectin (ED-A) or β-actin (Abcam, Cambridge, MA, USA). The bound primary antibodies were subsequently hybridized to horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgGs (Calbiochem, San Diego, CA, USA), and the immunoreactive bands were developed with a chemiluminescence kit (Perkin Elmer, Waltham, MA, USA). The optical densities of the bands for PPARδ (50 kDa), p-STAT3 (88 kDa), STAT3 (88 kDa), CCN2 (38 kDa), fibronectin (270 kDa), and β-actin (43 kDa) were quantified as described in our previous report [59 (link)].

Lo S.H., Hsu C.T., Niu H.S., Niu C.S., Cheng J.T, & Chen Z.C. (2017). Ginsenoside Rh2 Improves Cardiac Fibrosis via PPARδ–STAT3 Signaling in Type 1-Like Diabetic Rats. International Journal of Molecular Sciences, 18(7), 1364.

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Western Blot Analysis of SSEA-1 Expression

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At the end of treatment, the cells were lysed in the ice-cold whole cell extract buffer (pH7.4) containing 250 mM NaCl, 50 mM HEPES, and 0.1% NP-40. The protease inhibitors including 1 μg/ml aprotinin, 0.5 μg/ml leupeptin, and 100 μg/ml 4-(2-aminoethyl) benzenesulfonyl fluoride were added to the cell suspension. The lysate was vibrated for 30 min at 4°C and centrifuged at 12,000 rpm for 10 min. The protein concentrations were determined by the BCA protein assay kit (Pierce, Rockford, IL). Total protein extracts were prepared for western blot analysis using specific SSEA-1 and actin antibodies. Briefly, equal amounts of proteins in samples were subjected to electrophoresis using 10% sodium dodecyl sulfate-polyacrylamide gels. After electrophoretic transfer of proteins onto polyvinylidene fluoride membranes, they were sequentially hybridized with primary antibody and followed with a HRP-conjugated second antibody. Finally, the protein bands were visualized followed by detection with a chemiluminescence kit (PerkinElmer Life and Analytical Sciences, Boston, MA). To verify equal protein loading and transfer, actin was used as the protein loading control. The gel digitizing software, Un-Scan It gel (Ver.6.1, SilkScientific, Inc., Orem, UT), was used to analyze the intensity of bands in X-ray film.

Hsu T.C., Liu K.K., Chang H.C., Hwang E, & Chao J.I. (2014). Labeling of neuronal differentiation and neuron cells with biocompatible fluorescent nanodiamonds. Scientific Reports, 4, 5004.

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Cardiac Cell Protein Fractionation and Analysis

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Isolation of total protein extracts and cytosolic and nuclear protein fractions from AC16 cardiac cells or frozen tissue slides was performed as previously described.^{45 (link)} For immunoblotting, protein fractions were resolved by 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes. Proteins were identified by using several antibodies and a chemiluminescence kit (PerkinElmer, Waltham, MA, USA).
For coimmunoprecipitation studies, FOS antibody was first bound to Protein A/G Plus agarose beads by covalent crosslinking with dimethyl pimelimidate and following a standard method. Next, cell nuclear extracts (20 μg) were diluted with dilution buffer (10 mmol/L PBS, 50 mmol/L KCl, 0.05 mmol/L EDTA, 2.5 mmol/L MgCl₂, 8.5% glycerol, 1 mmol/L dithiothreitol, 0.1% Triton X-100, BSA 2%, and 1 mg/mL nonfat milk) and incubated on a rocker platform for 18 h at 4 °C with 100 μL Protein A/G Plus agarose beads containing 2 μg of bound FOS antibody. The agarose beads were centrifuged and washed with PBS. After centrifugation, the protein was eluted, and the supernatant was subjected to electrophoresis and immunoblotting with the anti-acetylated-lysine antibody.

Palomer X., Román-Azcona M.S., Pizarro-Delgado J., Planavila A., Villarroya F., Valenzuela-Alcaraz B., Crispi F., Sepúlveda-Martínez Á., Miguel-Escalada I., Ferrer J., Nistal J.F., García R., Davidson M.M., Barroso E, & Vázquez-Carrera M. (2020). SIRT3-mediated inhibition of FOS through histone H3 deacetylation prevents cardiac fibrosis and inflammation. Signal Transduction and Targeted Therapy, 5, 14.

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Western Blot Analysis of Synaptic Proteins

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Tissue corresponding to the NAc or CPu was collected from brains of each male or female rat (N = 6–9) rapidly after sacrifice. Following homogenization, 30–50 μg of protein from the indicated region were incubated in sample buffer for 3–5 min at 95 °C. Proteins were resolved by electrophoresis on 10% polyacrylamide gels under denaturing conditions (SDS–PAGE) and transferred onto nitrocellulose or PVDF membranes (Bio-Rad Laboratories, Hercules, CA) using a semidry transfer system (Invitrogen). Membranes were blocked in TBS-Tween (TBS-T)/5% nonfat milk for 1 h followed by incubation with PBS-T/5% nonfat milk containing the indicated first antibody overnight at 4 °C. Membranes were washed in TBS-T (3 × 10 min) and incubated with the appropriate horseradish peroxidase (HRP)-conjugated polyclonal secondary antibody (Bio-Rad) for 2 h at room temperature. After three washes as indicated above, signal detection was performed using a chemiluminescence kit (Perkin-Elmer). The primary antibodies used were anti-phosphoGSK-3 (1:1000, Cell Signaling), anti-BDNF (1:1000, Abcam), anti-phosphoTrkB (1:1000, Cell Signaling), and rabbit anti-GAPDH (1:10,000 or 1:20,000 Abcam).

Hasbi A., Nguyen T., Rahal H., Manduca J.D., Miksys S., Tyndale R.F., Madras B.K., Perreault M.L, & George S.R. (2020). Sex difference in dopamine D1-D2 receptor complex expression and signaling affects depression- and anxiety-like behaviors. Biology of Sex Differences, 11, 8.

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Western Blot Analysis of EMT Markers

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Total cell lysates were prepared using RIPA buffer supplemented with protease inhibitors (Halt Protease inhibitor cocktail, Thermo Scientific) and 40 μg/ml PMSF (Sigma). Cell extracts were resolved in 10% SDS–PAGE and transferred to PVDF membrane (PerkinElmer, Waltham, MA). The membranes were blocked by 5% skim milk in TBS with 0.1% of Tween-20 (TBST) for 2 h at room temperature prior to be incubated overnight with primary antibodies. The primary antibodies including E-cadherin (ab40772), N-cadherin (ab280375), Vimentin (ab92547), and ZEB1 (ab276129) were purchased from Abcam (USA). Phospho-STAT3 (Tyr705) (#9145) and STAT3 (#4904) were obtained from Cell Signaling Technology (USA). The membranes were than washed thrice in TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h. After consecutive washes, the membranes were visualized using a chemiluminescence kit (PerkinElmer, USA).

Liu Z., Ma L., Sun Y., Yu W, & Wang X. (2021). Targeting STAT3 signaling overcomes gefitinib resistance in non-small cell lung cancer. Cell Death & Disease, 12(6), 561.

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