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Chemiluminescent detection reagent

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Chemiluminescent detection reagent is a laboratory tool used to detect and quantify specific biomolecules, such as proteins or nucleic acids, in a sample. The reagent emits light upon a chemical reaction, which can be measured and analyzed to determine the presence and amount of the target substance.

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Lab products found in correlation

Nitrocellulose membrane, by Bio-Rad (3 mentions) Nupage bis tris sds page gel, by Thermo Fisher Scientific (2 mentions) Iblot 2 gel transfer device, by Thermo Fisher Scientific (2 mentions) Ibright fl1500, by Thermo Fisher Scientific (2 mentions) Protease inhibitor, by Roche (2 mentions) Hrp conjugated β actin antibody, by Merck Group (1 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions) Phospho gsk3β, by Cell Signaling Technology (1 mentions) Anti mouse and anti rabbit antibodies, by Jackson ImmunoResearch (1 mentions) Gsk3β, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Qproteome ffpe tissue kit, by Qiagen (1 mentions) Anti histone h3 antibody, by Abcam (1 mentions) Rabbit hrp conjugated secondary antibodies, by GE Healthcare (1 mentions) Rabbit monoclonal anti trimethyl histone h3 lys27 antibody, by Merck Group (1 mentions) Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions) Ab195975, by Abcam (1 mentions) Horseradish peroxidase conjugated anti rabbit antibody, by Jackson ImmunoResearch (1 mentions) Phosphatase inhibitor cocktail, by Merck Group (1 mentions) Cox 2, by Cell Signaling Technology (1 mentions)

16 protocols using chemiluminescent detection reagent

1

Immunoblotting of Macrophage Proteins

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BMDMs were lysed in 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0, with protease inhibitor cocktail (Roche) and resolved by SDS-PAGE using 4–12% or 12% NuPAGE Bis-Tris gels (Invitrogen). Proteins were transferred onto PVDF membranes and probed with rabbit polyclonal antibodies against caspase-1 (a gift from Gabriel Nuñez), YopM (a gift from Sue Straley), pyrin (Chae et al., 2003 (link)), or phospho-GSK-3β (Cell Signaling), a rabbit monoclonal antibody against GSK-3β (Cell Signaling), and mouse monoclonal anti-YopE 202.19 and 149.27 antibodies (unpublished data). Anti-rabbit and anti-mouse antibodies (Jackson ImmunoResearch) conjugated to HRP were used as secondary reagents. An HRP-conjugated β-actin antibody (Sigma) was used to control for loading. Proteins were visualized using chemiluminescent detection reagent (GE Healthcare).
Chung L.K., Park Y.H., Zheng Y., Brodsky I.E., Hearing P., Kastner D.L., Chae J.J, & Bliska J.B. (2016). The Yersinia Virulence Factor YopM Hijacks Host Kinases to Inhibit Type III Effector-Triggered Activation of the Pyrin Inflammasome. Cell host & microbe, 20(3), 296-306.
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2

Histone H3 Immunoblotting for FFPE Samples

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Cell lysate was extracted from FFPE tissue samples using a Qproteome FFPE Tissue Kit (Qiagen). 15 μg of total protein from each sample was run on 4–12% bis-tris precast polyacrylamide gel (Thermo Fisher Scientific), transferred to PVDF membrane using a wet system and probed overnight with primary antibody. The following primary antibodies were used: rabbit polyclonal anti-Histone H3 antibody (Abcam, Cambridge, MA), rabbit monoclonal anti-histone H3 K27M (RevMAb Biosciences) and rabbit monoclonal anti-trimethyl-histone H3 (Lys27) antibody (Merck Millipore). The blot was then probed with the rabbit HRP-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK). Membranes were revealed using chemiluminescent detection reagent (GE Healthcare) and enhanced signal was detected in CCD imaging system. Protein extracted from the FFPE tumor sample of H3F3A wild-type glioma (IDH1 mutated) case was used as a negative control for H3 K27M and as a positive control for H3K27me3 [19 (link)].
Maeda S., Ohka F., Okuno Y., Aoki K., Motomura K., Takeuchi K., Kusakari H., Yanagisawa N., Sato S., Yamaguchi J., Tanahashi K., Hirano M., Kato A., Shimizu H., Kitano Y., Yamazaki S., Yamashita S., Takeshima H., Shinjo K., Kondo Y., Wakabayashi T, & Natsume A. (2020). H3F3A mutant allele specific imbalance in an aggressive subtype of diffuse midline glioma, H3 K27M-mutant. Acta Neuropathologica Communications, 8, 8.
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3

Western Blot Analysis of Inflammatory Proteins

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Five to ten micrograms of protein from the cell lysates was run on 4 to 12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen by ThermoFisher Scientific) and transferred to polyvinylidene difluoride membranes (ThermoFisher Scientific) using an iBlot 2 gel transfer device (Life Technologies). Membranes were blocked in 5% nonfat dairy milk and incubated with primary antibodies overnight. The primary antibodies used are rabbit-anti-mouse monoclonal total pyrin antibody (ab195975; abcam), rabbit-anti-mouse monoclonal antibody phospho-serine 205 (ab201784; abcam), rabbit-anti-mouse/human IL-1β (number 12242; Cell Signaling), and rabbit-anti-mouse/human polyclonal β-actin (number 4967; Cell Signaling). Horseradish peroxidase-conjugated anti-rabbit antibody (Jackson Laboratory) was used as a secondary antibody. Proteins were visualized using chemiluminescent detection reagent (GE Healthcare) on an iBright FL1500 (ThermoFisher Scientific).
Loeven N.A., Perault A.I., Cotter P.A., Hodges C.A., Schwartzman J.D., Hampton T.H, & Bliska J.B. (2021). The Burkholderia cenocepacia Type VI Secretion System Effector TecA Is a Virulence Factor in Mouse Models of Lung Infection. mBio, 12(5), e02098-21.
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4

Immunoblotting Protein Expression Analysis

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Cells were washed twice with 1x cold phosphate-buffered saline (PBS) and lysed in RIPA buffer: 0.01 M Tris, pH 7.4, 0.15 M NaCl, 0.001 M EDTA, 0.001 M EGTA, 1% Triton X-100, phosphatase inhibitor cocktail (Sigma Aldrich), and protease inhibitor (Roche, Rotkreuz, Switzerland). The lysates were cleared by centrifugation for 15 min at 17,000 g. Protein concentration was measured by BCA assay (Pierce Biotechnology, IL, USA). Equal amounts of protein (30 μg) were separated by SDS-PAGE and immunoblotted with specific primary antibodies overnight at 4 °C. 1:1000 Anti-c-jun (cat. no. 60A8, Cell Signaling Technologies, Berkeley, CA, USA), 1:1000 p65 (cat. no. D14E12, Cell Signaling Technologies), 1:1000 COX-2 (cat. no. 4842S, Cell Signaling Technologies), 1:10000 anti-actin (A5441, Sigma Aldrich, USA), and 1:3000 anti-TonEBP antibody [7 (link)] were used. 1:10000 HRP-conjugated mouse or rabbit secondary antibodies were used for detection. Antigen-antibody binding was detected by chemiluminescent detection reagent (GE Healthcare, NJ, USA).
Jeong G.W., Lee H.H., Lee-Kwon W, & Kwon H.M. (2020). Microglial TonEBP mediates LPS-induced inflammation and memory loss as transcriptional cofactor for NF-κB and AP-1. Journal of Neuroinflammation, 17, 372.
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5

Notch Signaling Pathway Protein Analysis

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Whole cell lysates were prepared from frozen xenograft samples or cell lines using Mammalian Protein Extraction Reagent (Thermo Scientific) lysis buffer supplemented with inhibitors of endogenous protease, kinase, and phosphatase activity (all obtained from Sigma-Aldrich). Twenty micrograms of protein from each sample were separated on 7.5 or 10% polyacrylamide gels and transferred to a PVDF membrane. Following transfer, membranes were blocked with 5% milk in 1× TBS, 0.1% Tween-20 (TBST) and incubated in diluted (1:1000) primary antibody overnight, according to the manufacturer’s recommendations. Primary antibodies used were a rabbit monoclonal anti-Notch1 antibody, a rabbit monoclonal anti-cleaved Notch1 (Val1744) antibody, and a rabbit monoclonal anti-Notch3 antibody (Cell Signaling). Membranes were then incubated with a horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) and developed using a chemiluminescent detection reagent obtained from GE Healthcare Life Sciences. Equivalent protein loading was verified by stripping the blots and re-probing with a mouse anti-Pan-Actin antibody (NeoMarkers).
Groeneweg J.W., DiGloria C.M., Yuan J., Richardson W.S., Growdon W.B., Sathyanarayanan S., Foster R, & Rueda B.R. (2014). Inhibition of Notch Signaling in Combination with Paclitaxel Reduces Platinum-Resistant Ovarian Tumor Growth. Frontiers in Oncology, 4, 171.
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6

Protein Analysis of Cell Lysates

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Cell lysates were run on 4–12% NuPAGE Bis-Tris SDS-PAGE gels (Invitrogen by ThermoFisher Scientific) and transferred to PVDF membranes (ThermoFisher Scientific) using an iBlot 2 Gel Transfer Device (Life Technologies). Membranes were blocked in 5% non-fat dairy milk and incubated with primary antibody overnight. The primary antibodies used were rabbit MAb for GSDMD (Abcam, ab209845), MAb for Histone 3 (Cell Signaling #96C10), Mab for Citrullinated Histone 3 (Abcam #ab5103) and rabbit polyclonal for b-actin (Cell Signaling, #4967), and polyclonal ab for ExoS (from Arne Rietsch). HRP-conjugated anti-rabbit (Jackson Immuno Research) or HRP-conjugated anti-mouse (Jackson Immuno Research) was used as a secondary antibody. Protein bands reacting with antibodies were visualized using chemiluminescent detection reagent (GE Healthcare) on an iBright FL1500 (ThermoFisher Scientific).
Reuven A.D., Mwaura B.W, & Bliska J.B. (2024). ExoS Effector in Pseudomonas aeruginosa Hyperactive Type III Secretion System Mutant Promotes Enhanced Plasma Membrane Rupture in Neutrophils. bioRxiv.
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7

Quantitative Protein Analysis in Cell Lysates

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Cells were washed two times with cold PBS and lysed in RIPA buffer (0.01 M Tris, pH 7.4, 0.15 M NaCl, 0.001 M EDTA, 0.001 M EGTA, 1% Triton-X 100, 0.002 M PMSF, and protease inhibitor (Roche, Rotkreuz, Switzerland)). After centrifugation of lysate, supernatant was used for immunoblot assay. Protein concentration was measured by BCA protein assay system (Pierce Biotechnology, IL, USA). Equal amounts of protein from each sample were separated by SDS-PAGE. Anti-Adrb3 (Abcam, Cambridge, UK, #ab59685), anti-UCP-1 (EMD Milipore, CA, USA, #AB1426), anti-pAKT (Cell signaling, #9271 S), anti-AKT (Cell signaling, #9272), anti-Hsc70 (Rockland, PA, USA, #200-301-A28), and anti-TonEBP antibody5 (link) were used for immunoblotting at 1:1000 dilution. HRP-conjugated mouse, rabbit, or goat secondary antibodies were used for detection. The antigen–antibody binding was detected by chemiluminescent detection reagent (GE Healthcare, NJ, USA). Uncropped western blots are shown in Supplementary Fig. 12.
Lee H.H., An S.M., Ye B.J., Lee J.H., Yoo E.J., Jeong G.W., Kang H.J., Alfadda A.A., Lim S.W., Park J., Lee-Kwon W., Kim J.B., Choi S.Y, & Kwon H.M. (2019). TonEBP/NFAT5 promotes obesity and insulin resistance by epigenetic suppression of white adipose tissue beiging. Nature Communications, 10, 3536.
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8

Western Blot and Coimmunoprecipitation

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Cells were washed in ice-cold PBS following by lysis with RIPA buffer. Equal amounts of protein samples were boiled in Laemmli buffer (Bio-Rad) for 5 minutes at 95 C. Samples were subjected to SDS-PAGE separation and transferred to polyvinylidene difluoride membrane (Bio-Rad). The membranes were blocked in 5% BSA in TBST (TBS þ 0.1% Tween-20) at room temperature for 1 hour, followed by incubation of primary antibodies overnight at 1:2,000 dilution and 1-hour incubation at with respective horseradish peroxidase-labeled secondary antibody at 1:5,000 dilution. Bands were visualized with chemiluminescent detection reagent (GE Healthcare, RPN2106). Coimmunoprecipitation was performed with 500 mg of nuclear lysate incubated overnight with respective antibodies. Dynabeads Protein G (Invitrogen) was used to harvest the immunoprecipitates. Twopercent input was used as a loading control. Antibodies used are listed in Supplementary Methods.
Chong P.S.Y., Chooi J.Y., Lim J.S.L., Toh S.H.M., Tan T.Z, & Chng W.J. (2021). SMARCA2 Is a Novel Interactor of NSD2 and Regulates Prometastatic PTP4A3 through Chromatin Remodeling in t(4;14) Multiple Myeloma. Cancer research, 81(9).
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9

Quantification of Oxidative Protein Damage

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The levels of the oxidative protein damage marker, protein carbonyl, were measured in nuclear, mitochondrial, and cytosolic fractions using the OxyBlot Protein Oxidation Detection Kit (MilliporeSigma) according to the manufacturer’s instructions. In brief, 8 μg of protein in each fraction was added to a microcentrifuge tube, denatured by adding 1 volume of 12% SDS, and derivatized by adding 2 volumes of the 1x DNPH (2,4-dinitrophenylhydrazine) Solution. The tube was incubated for 15 min at room temperature and the reaction was stopped by adding 1.5 volume of the Neutralization Solution. The protein sample was reduced by treatment with 0.74 M 2-mercaptoethanol, fractionated by SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad Laboratories). The membranes were incubated with the anti-DNP (2,4-dinitrophenyl) antibody followed by the horseradish peroxidase-conjugated secondary antibody. Chemiluminescent detection reagents (GE Healthcare Life Sciences) were used to visualize proteins. Band intensities were quantified using ImageJ software (National Institutes of Health).
Kim M.J., Han C., White K., Park H.J., Ding D., Boyd K., Rothenberger C., Bose U., Carmichael P., Linser P.J., Tanokura M., Salvi R, & Someya S. (2020). Txn2 haplodeficiency does not affect cochlear antioxidant defenses or accelerate the progression of cochlear cell loss or hearing loss across the lifespan. Experimental gerontology, 141, 111078.
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10

Cytokine Profiling of Lavage Fluid

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Lavage fluid supernatant was concentrated 10-fold on Centricon filters (Millipore, Billerica, MA). The concentrated supernatant was assessed for 36 cytokines with a Human Cytokine Array Panel A (R&D Systems, Minneapolis, MN). The supernatant was mixed with a cocktail of biotinylated detection antibodies and incubated on a nitrocellulose membrane, embedded with cognate capture antibodies. Arrays were then treated with streptavidin-horseradish peroxidase for 30 min. Chemiluminescent detection reagents were added (GE Healthcare, Piscataway, NJ) and the array was developed. The signal present was proportional to the amount of cytokine captured. Optical densities of obtained values were measured and quantified using Image J software.
Barjaktarevic I.Z., Crystal R.G, & Kaner R.J. (2016). The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+ Smokers. Journal of Immunology Research, 2016, 3463104.
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