Immobiliontm western chemiluminescent hrp substrate

Manufactured by Merck Group

Sourced in United States, Canada

ImmobilionTM Western Chemiluminescent HRP Substrate is a laboratory reagent used in Western blot analysis. It is designed to detect and quantify target proteins that have been labeled with horseradish peroxidase (HRP) conjugated antibodies. The substrate emits a chemiluminescent signal upon reaction with HRP, which can be captured and analyzed using appropriate imaging equipment.

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6 protocols using immobiliontm western chemiluminescent hrp substrate

Western Blot Protein Analysis Protocol

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Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [41 (link)]. Antibodies against p-Rb^ser780, p-Rb^{ser807/ser811}, Rb, Cyclin D1, p16^INK4a, p-CDK6, CDK6, p-AKT^ser473, AKT, pEGFR^tyr1068, EGFR, p53, p-MDM2, p21, c-Myc, actin, and HRP-conjugated secondary antibodies was from Cell Signaling Technology; the chemiluminescence system (Immobilion^TM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA, USA). Reagents for electrophoresis and blotting analysis were from BIO-RAD (Hercules, CA, USA). The whole Western blots are shown in Figures S3–S5.

La Monica S., Fumarola C., Cretella D., Bonelli M., Minari R., Cavazzoni A., Digiacomo G., Galetti M., Volta F., Mancini M., Petronini P.G., Tiseo M, & Alfieri R. (2020). Efficacy of the CDK4/6 Dual Inhibitor Abemaciclib in EGFR-Mutated NSCLC Cell Lines with Different Resistance Mechanisms to Osimertinib. Cancers, 13(1), 6.

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Protein Extraction, Solubilization, and Analysis

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Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [13 (link)]. Antibody against Thymidylate Synthase (TS) was from Upstate (Lake Placid, NY); antibody against MET, p-EGFRTyr1068, EGFR, pAKTser473, AKT, p-ERK1/2Thr202/Tyr204, ERK1/2, Bim, cleaved caspase-7, PARP and HRP-conjugated secondary antibodies was from Cell Signaling Technology (Beverly, MA); antibody against Actin was from Sigma; and chemoluminescence system (ImmobilionTM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA). Reagents for electrophoresis and blotting analysis were from BIO-RAD (Hercules, CA).

La Monica S., Minari R., Cretella D., Flammini L., Fumarola C., Bonelli M., Cavazzoni A., Digiacomo G., Galetti M., Madeddu D., Falco A., Lagrasta C.A., Squadrilli A., Barocelli E., Romanel A., Quaini F., Petronini P.G., Tiseo M, & Alfieri R. (2019). Third generation EGFR inhibitor osimertinib combined with pemetrexed or cisplatin exerts long-lasting anti-tumor effect in EGFR-mutated pre-clinical models of NSCLC. Journal of Experimental & Clinical Cancer Research : CR, 38, 222.

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Protein Analysis by 1-D PAGE

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Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [22 (link)]. Antibodies against p-FGFR^Tyr653/654, FGFR1, p-FRS2-α^Tyr196, p-ERK1/2^{Thr202/Tyr204}, ERK1/2, p-mTOR^Ser2448, mTOR, p-AKT^ser473, AKT, p-P70S6K^Thr389, P70S6K, p-AMPKα1^Thr172, p-src^Tyr416, src, p-FAK^Tyr397, FAK were from Cell Signaling Technology (Beverly, MA); the antibodies against GLUT-1 and AMPKα1 were from AbCam (Cambridge, MA); the antibody against HIF-1α was from BD Biosciences (Franklin Lakes, NJ); the antibody against actin was from Sigma-Aldrich. HRP-conjugated secondary antibodies were from Pierce (Rockford, IL) and chemiluminescence system (ImmobilionTM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA).

Fumarola C., Cretella D., La Monica S., Bonelli M.A., Alfieri R., Caffarra C., Quaini F., Madeddu D., Falco A., Cavazzoni A., Digiacomo G., Mazzaschi G., Vivo V., Barocelli E., Tiseo M., Petronini P.G, & Ardizzoni A. (2017). Enhancement of the anti-tumor activity of FGFR1 inhibition in squamous cell lung cancer by targeting downstream signaling involved in glucose metabolism. Oncotarget, 8(54), 91841-91859.

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Protein Analysis by Western Blotting

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Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere (25 (link)). Antibodies against p-FGFR^Tyr653/654, FGFR1, p-ERK1/2^{Thr202/Tyr204}, ERK1/2, p-mTOR^Ser2448, mTOR, p-AKT^ser473, AKT, p-P70S6K^Thr389, P70S6K were from Cell Signaling Technology (Beverly, MA); the antibody against actin was from Sigma-Aldrich. HRP-conjugated secondary antibodies were from Pierce (Rockford, IL) and chemiluminescence system (ImmobilionTM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA). The chemiluminescent signal was acquired by C-DiGit® Blot Scanner (LI-COR Biotechnology, Lincoln, NE). Where indicated, phosphorylation levels were quantified by Image Studio™ Software (LI-COR Biotechnology), and normalized to the corresponding protein levels.

Fumarola C., Bozza N., Castelli R., Ferlenghi F., Marseglia G., Lodola A., Bonelli M., La Monica S., Cretella D., Alfieri R., Minari R., Galetti M., Tiseo M., Ardizzoni A., Mor M, & Petronini P.G. (2019). Expanding the Arsenal of FGFR Inhibitors: A Novel Chloroacetamide Derivative as a New Irreversible Agent With Anti-proliferative Activity Against FGFR1-Amplified Lung Cancer Cell Lines. Frontiers in Oncology, 9, 179.

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Quantitative Western Blot Analysis

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Western blot was conducted as described previously^{50 (link)}. Antibodies against cleaved Caspase-3 (9661; 1:1000), BIM (2933; 1:1000), MCL-1 (94296; 1:1000), actin (3700; 1:1000), and HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), GCS (12869-1-AP; 1:1000) was from Proteintech (Rosemont, IL, USA); the chemiluminescence system (ImmobilionTM Western Chemiluminescent HRP Substrate) was from Millipore. Reagents for electrophoresis and blotting analysis were from BIO-RAD (Hercules, CA, USA). The chemiluminescent signal was acquired by C-DiGit^® Blot Scanner and the spots were quantified by Image Studio™ Software, LI-COR Biotechnology (Lincoln, NE). The blots were cut prior to hybridization with antibodies during blotting. The original blots and replicates are available in supplementary information.

La Monica S., Vacondio F., Eltayeb K., Lodola A., Volta F., Viglioli M., Ferlenghi F., Galvani F., Galetti M., Bonelli M., Fumarola C., Cavazzoni A., Flammini L., Verzè M., Minari R., Petronini P.G., Tiseo M., Mor M, & Alfieri R. (2024). Targeting glucosylceramide synthase induces antiproliferative and proapoptotic effects in osimertinib-resistant NSCLC cell models. Scientific Reports, 14, 6491.

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Protein Extraction and Analysis

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Procedures for protein extraction and protein analysis by 1-D PAGE are described elsewhere [40 (link)]. Antibodies against PTEN, p-mTOR (Ser2448), m-TOR, p-AKT (Thr308), p-AKT (Ser473), AKT, P110α-PI3K, P110β-PI3K, p-P85-PI3K (Tyr458), p-FAK (Tyr925), FAK, p-GSK3 α/β (Ser21/9), GSK3 α/β, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, Rac1 and CDC42 were from Cell Signaling Technology (Beverly, MA). Antibody directed to p-FAK (Tyr397) was from Thermo Fisher Scientific.
The antibody against Actin was from Sigma–Aldrich (St Louis, MO). HRP-conjugated secondary antibodies were from Pierce (Rockford, IL) and chemiluminescence system (ImmobilionTM Western Chemiluminescent HRP Substrate) was from Millipore (Temecula, CA). The chemiluminescent signal was acquired by C-DiGit^® Blot Scanner and the spots were quantified by Image Studio^™ Software, LI-COR Biotechnology (Lincoln, NE).
Active GTP-bound RhoA family proteins were evaluated by using Active Rho family Detection Kit from Cell Signaling Technology, accordingly to manufactures instructions [15 (link)].

Cavazzoni A., La Monica S., Alfieri R., Ravelli A., Van Der Steen N., Sciarrillo R., Madeddu D., Lagrasta C.A., Quaini F., Bonelli M., Fumarola C., Cretella D., Digiacomo G., Tiseo M., Peters G.J., Ardizzoni A., Petronini P.G, & Giovannetti E. (2017). Enhanced efficacy of AKT and FAK kinase combined inhibition in squamous cell lung carcinomas with stable reduction in PTEN. Oncotarget, 8(32), 53068-53083.

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