Chemiluminescent detection substrate

Manufactured by GE Healthcare

Sourced in United States

Chemiluminescent detection substrate is a laboratory reagent used to detect and quantify specific biomolecules, such as proteins, in biological samples. It generates a luminescent signal in the presence of the target analyte, allowing for sensitive and accurate detection.

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Lab products found in correlation

Protease and phosphatase inhibitor tablet, by Roche (2 mentions) Ipvh00010, by Merck Group (2 mentions) Alx 803 335 c100, by Enzo Life Sciences (1 mentions) Phospho jnk, by Thermo Fisher Scientific (1 mentions) Ciap1, by Enzo Life Sciences (1 mentions) Ripk3, by Enzo Life Sciences (1 mentions) β actin, by Santa Cruz Biotechnology (1 mentions) Ripk1, by BD (1 mentions) Phosstop phosphatase inhibitor, by Roche (1 mentions) Hrp coupled secondary antibodies, by GE Healthcare (1 mentions) Immobilon p pvdf membrane, by Merck Group (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Complete protease inhibitor, by Roche (1 mentions) Immobilon p polyvinylidene difluoride membrane, by Merck Group (1 mentions) Anti p21 c 19, by Santa Cruz Biotechnology (1 mentions) Anti cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions) Anti phospho jnk, by Thermo Fisher Scientific (1 mentions) Anti iκbα, by Santa Cruz Biotechnology (1 mentions) Anti p53 1c12, by Cell Signaling Technology (1 mentions) Anti tubulin, by Merck Group (1 mentions)

4 protocols using chemiluminescent detection substrate

Isolation and Analysis of Intestinal Epithelial Cells

Cited 1 time

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IECs were isolated by sequential incubation of intestinal tissue in 1 mM dithiothreitol (DTT) and 1.5 mM EDTA solutions as described previously^{40 (link)}. Cell lysates from IECs and keratinocytes were prepared as described^{41 (link),42 (link)}. Lysis buffer was supplemented with protease and phosphatase inhibitor tablets (Roche). Cell lysates were separated on SDS–PAGE and transferred to PVDF membranes (IPVH00010, Millipore). Membranes were probed with primary antibodies against the following proteins: RIPK1 (610459, BD Biosciences), RIPK3 (ADI-905-242-100), cIAP1 (ALX-803-335-C100, Enzo), IκBα (sc-371), TRAF2 (sc-876), TRAF3 (sc-949), TRADD (sc-7868), p65 (sc-372), c-Rel (sc-71),HDAC1 (sc-7872), β-actin, (sc-1616, Santacruz), phospho-p65 (3033), JNK (9252), RelB (4922), phospho-p38 (9211), p38 (8690), phospho-ERK (9101), ERK (9102, Cell Signaling), phospho-JNK (44-682G, Invitrogen), p100/52 (NR1495), p105/50 (NR1157, NCI BRB), α-tubulin (T6074, Sigma), TRAF6 (597, MBL), c-FLIP (AG-20B-0005-C100, Adipogen), followed by secondary HRP-coupled antibodies (GE Healthcare and Jackson ImmuneResearch) and developed with chemiluminescent detection substrate (GE Healthcare and Thermo Scientific).

Dannappel M., Vlantis K., Kumari S., Polykratis A., Kim C., Wachsmuth L., Eftychi C., Lin J., Corona T., Hermance N., Zelic M., Kirsch P., Basic M., Bleich A., Kelliher M, & Pasparakis M. (2014). RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature, 513(7516), 90-94.

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Detailed Immunoblotting Workflow for Liver Proteins

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Protein extracts from primary hepatocytes or liver tissues were prepared in a standard lysis buffer containing 20 mM HEPES-KOH pH 7.6, 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 1% Triton X-100, 10% glycerol, phosSTOP phosphatase inhibitors (Roche, Basel, Switzerland) and complete protease inhibitors (Roche). Lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to Immobilon-P PVDF membranes (Millipore, Burlington, MA, USA), and analyzed by immunoblotting. Membranes were probed with primary antibodies against the following proteins: NEMO, p62, phospho-p62 (S351), S 6, phospho-S6, AMPK, phospho-AMPK, IKK2, LC3B, ATG16L1, α-Tubulin, actin and GAPDH, which were obtained from the suppliers mentioned in the antibody table. Membranes were then incubated either with secondary HRP-coupled antibodies (GE Healthcare, Chicago, IL, USA and Jackson ImmunoResearch, West Grove, PA, USA) and developed with chemiluminescent detection substrate (GE Healthcare) or with secondary antibodies coupled to IRDye680 or 800 and visualized with Odyssey infrared imaging system (Licor, Lincoln, NE, USA). Protein expression in immunoblots was performed by band intensity quantification using ImageJ (Version 1.53a; NIH, Bethesda, MD, USA) [42 (link)].

Kondylis V., Schneider F., Schorn F., Oikonomou N., Straub B.K., Werner S., Rosenstiel P, & Pasparakis M. (2022). p62 Promotes Survival and Hepatocarcinogenesis in Mice with Liver-Specific NEMO Ablation. Cancers, 14(10), 2436.

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Western Blot Analysis of Cellular Proteins

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Total protein cell extracts were separated by SDS–polyacrylamide gel electrophoresis gels and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore). Membranes were probed with primary antibodies; anti-p53(1C12) (Cell Signaling; 2524; 1:750 dilution), anti-p21(C-19) (Santa Cruz Biotechnology; sc-397; 1:1,000 dilution), anti-cleaved caspase-3 (Asp175) (Cell Signaling; 9661; 1:1,000 dilution), anti-phospho JNK (Invitrogen; 44-682G; 1:1,000 dilution), anti-JNK (Cell Signaling; 9252; 1:1,000 dilution), anti-IκBα (Santa cruz; sc-371; 1:1,000 dilution), anti-tubulin (Sigma; T6074; 1:5,000) and anti-actin (Santa Cruz Biotechnology; sc-1616; 1:1,000 dilution) antibodies at 4° O/N. Membranes were incubated with secondary horseradish peroxidase-coupled antibodies (GE Healthcare and Jackson Immune Research) and developed with chemiluminescent detection substrate (GE Healthcare and Thermo Scientific).

Fernández-Majada V., Welz P.S., Ermolaeva M.A., Schell M., Adam A., Dietlein F., Komander D., Büttner R., Thomas R.K., Schumacher B, & Pasparakis M. (2016). The tumour suppressor CYLD regulates the p53 DNA damage response. Nature Communications, 7, 12508.

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Isolation and Analysis of Intestinal Epithelial Cells

Cited 1 time

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IECs were isolated by subsequent incubation of intestinal tissue in 1 mM dithiothreitol (DTT) and 1.5 mM EDTA solutions as described previously (Dannappel et al., 2014 (link)). Cell lysis buffer was supplemented with protease and phosphatase inhibitor tablets (Roche). Cell lysates were separated on SDS-PAGE and transferred to PVDF membranes (IPVH00010, Millipore). Primary antibodies used for immunoblot analysis: RelA C-20 (sc-372), RelB C-19 (sc-226), and c-Rel (sc-71), β-actin 1-19 (sc-161) and HDAC1 H-51 (sc-7872) from Santa Cruz, TNFR1 (D317K); Caspase 3 (9662), cleaved Caspase 3 (9661) from Cell Signaling; RIPK1 (610459) from BD; RIPK3 (ADI-905-242-100) from Enzo Life Sciences; FADD (1F7) 05-486 from Upstate; Cre 69050-3 from Novagen; Caspase-8 (ALX-804-447) from Alexis; and NEMO (homemade rabbit polyclonal serum) and α-tubulin (T6074) from Sigma. Secondary HRP-coupled antibodies (GE Healthcare, Jackson Immuno Research) and chemiluminescent detection substrate (GE Healthcare and Thermo Scientific) were used. For immunoprecipitation, colon tissue was lysed in IP buffer and FADD was precipitated with goat anti-FADD (M19) (sc-6036) antibody from Santa Cruz linked to protein G Dynabeads (Life Technologies). After extensive washes, beads were boiled in SDS-containing sample buffer.

Vlantis K., Wullaert A., Polykratis A., Kondylis V., Dannappel M., Schwarzer R., Welz P., Corona T., Walczak H., Weih F., Klein U., Kelliher M, & Pasparakis M. (2016). NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-κB-Dependent and -Independent Functions. Immunity, 44(3), 553-567.

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