Immobilon western detection kit

Manufactured by Merck Group

Sourced in United States

The Immobilon Western Detection kit is a laboratory equipment product designed for the detection and visualization of proteins in Western blot analysis. It provides the necessary reagents and components to enable the chemiluminescent detection of target proteins on a membrane.

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Lab products found in correlation

Ec3 imaging system, by Analytik Jena (3 mentions) Anti gapdh antibody, by Merck Group (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Immobilon p transfer membrane, by Merck Group (1 mentions) Trans blot semi dry transfer cell, by Bio-Rad (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Anti mmp 13, by Santa Cruz Biotechnology (1 mentions) Anti nos2, by Cell Signaling Technology (1 mentions) Anti pgrn, by Santa Cruz Biotechnology (1 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Anti vcam 1, by Cell Signaling Technology (1 mentions) Anti cox 2, by Cell Signaling Technology (1 mentions) Complete mini, by Roche (1 mentions) Phosstop, by Roche (1 mentions) α tubulin, by Abcam (1 mentions) Protease inhibitor, by Roche (1 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions) A1978, by Merck Group (1 mentions) In vivo imaging system fx pro, by Carestream (1 mentions) Apr 004, by Alomone (1 mentions)

Spelling variants of this product

Immobilon (435 citations) Immobilon Western (255 citations) Immobilon Western kit (70 citations) Immobilon kit (10 citations) Immobilon ECL Western Blotting Detection Kit Reagent (3 citations) Immobilon Western blotting detection kit (2 citations) Immobilon Western Chemiluminescent HRP detection kit (2 citations) ImmobilonTM Western enhanced chemiluminescence detection kit (2 citations)

7 protocols using immobilon western detection kit

Protein Extraction and Western Blotting

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Small pieces of frozen tissues were placed into 1.5 mL centrifuge tubes and then homogenized by using a Turrax homogenizer (IKA, Germany) in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orhtovanadate, 0.5% Triton X-100, 1mM PMSF, protease inhibitor cocktail). Tissues lysates were obtained by centrifugation at 14.000g for 20 min at 4°C.
Equal amount of protein were subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride transfer membrane (Immobilon-P transfer membrane, Millipore, MA) using a Trans-Blot semi-dry transfer cell (BioRad, CA, USA). Blots were incubated with the appropriate antibody (anti-SERPINE2 1:1000, R&D Systems, MN, USA; anti-ITIH5 1:1000, ABCAM, UK; anti-WISP2 1:1000, Abnova, Taiwan). Immunoblots were visualized with Immobilon Western Detection kit (Millipore, MA) using horseradish peroxidase-labeled secondary antibody. To confirm equal loading for each sample, membranes were incubated with stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-β-actin antibody (Sigma, MO, USA). Images were captured and analyzed with an EC3 imaging system (UVP, CA, USA).

Conde J., Scotece M., Abella V., Gómez R., López V., Villar R., Hermida M., Pino J., Gómez-Reino J.J, & Gualillo O. (2015). Identification of Novel Adipokines in the Joint. Differential Expression in Healthy and Osteoarthritis Tissues. PLoS ONE, 10(4), e0123601.

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Protein Extraction and Western Blot Analysis

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After the cell treatment, cells were rapidly washed with ice-cold phosphate buffered saline and scraped in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM Sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% Triton X-100, 1 mM PMSF and protease inhibitor cocktail from Thermo Scientific). Lysed cells were centrifuged at 14.000 g for 20 min. SDS-PAGE and blotting procedure were carried on as previously described38 (link). Immunoblots were incubated with the appropriate antibody (anti-PGRN diluted 1:1000, Santa Cruz, CA, USA; anti-NOS2 diluted 1:1000, Cell Signalling, MA, USA; anti-COX2 diluted 1:50, anti-VCAM-1 diluted 1:1000, Cell Signalling, MA, USA; anti-MMP13 diluted 1:500, Santa Cruz, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore, MA) using anti-rabbit (GE Healthcare, UK) horseradish-peroxidise-labelled secondary antibody diluted 1:2000. To confirm equal loading in each sample, the membranes were stripped in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-GAPDH antibody diluted 1:30000 (Sigma, MO, USA). The images were captured and analyzed with an EC3 imaging system (UVP). Densitrometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Abella V., Scotece M., Conde J., López V., Pirozzi C., Pino J., Gómez R., Lago F., González-Gay M.Á, & Gualillo O. (2016). The novel adipokine progranulin counteracts IL-1 and TLR4-driven inflammatory response in human and murine chondrocytes via TNFR1. Scientific Reports, 6, 20356.

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Protein Extraction and Western Blotting

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To obtain a total homogenate, frozen heart samples were homogenized in ice-cold buffer containing 20 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerophosphate, 1 mmol/L Na₃VO₄, 1 mg/mL leupeptin, 50 μg/mL phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor cocktail (Complete mini, Roche Molecular Biochemicals, Mannheim, Germany), and a phosphatase inhibitor cocktail (PhosSTOP, Roche Molecular Biochemicals, Mannheim, Germany). The homogenate was centrifuged at 13000g for 15 minutes to obtain the supernatant. Protein concentration was determined using the Bradford assay.
Equal amounts of proteins (80 mg) were electrophoresed on 4% to 12% polyacrylamide gels and then blotted onto PVDF membranes (Millipore, Bedford, Massachusetts). After blocking had been performed with a tris-buffered saline (TBS-T) buffer containing 5% bovine serum albumin (BSA), the blots were incubated with antibodies that recognize the following: p53 (1C12, Cell Signaling Technology, Beverly, Massachusetts), p53 (PAb122, BD Biosciences, San Jose, California), and α-tubulin (Abcam, Cambridge, Massachusetts). Immunoblotted proteins were visualized using an Immobilon Western Detection Kit (Millipore, Billerica, Massachusetts).

Yano T., Abe K., Tanno M., Miki T., Kuno A., Miura T, & Steenbergen C. (2018). Does p53 Inhibition Suppress Myocardial Ischemia–Reperfusion Injury?. Journal of cardiovascular pharmacology and therapeutics, 23(4), 350-357.

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Western Blot Analysis of P2X7R and Panx1

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Tissue samples were sonicated in lysis buffer (5 mM EDTA, 1 mM sodium orthovanadate, 1 mM NaHCO₃, 2 mM PMSF [Sigma-Aldrich], and 1× protease inhibitor [Roche, Mannheim, Germany]), electrophoresed on 10% SDS-PAGE gels for separation, and then transferred to nitrocellulose membranes (Whatman GmbH, Dassel, Germany). The membranes were probed with primary polyclonal antibodies to P2X7R (1:1000; APR-004, Alomone Labs, Israel), Panx1 (N [Term], 1:100; Cat 487900, Invitrogen Corporation, CA), and β-actin (1:35000; A1978, Sigma-Aldrich), followed by incubation with the respective horseradish peroxidase (HP)-conjugated anti-rabbit IgG or anti-mouse IgG (1:10000; Santa Cruz Biotechnology, TX) antibody. The protein bands were detected with the In Vivo FX PRO imaging system (Carestream, NY) using the Immobilon western detection kit (Millipore, MA), as previously described.^{7 (link)} Densitometric analyses were performed using ImageJ (NIH) software. Measured intensities for all samples were first normalized with the respective β-actin loading control, and then with either the age-matched non-loaded WT or the age-matched non-loaded Panx1^−/− bone tissue samples.

Seref-Ferlengez Z., Urban-Maldonado M., Sun H.B., Schaffler M.B., Suadicani S.O, & Thi M.M. (2018). Role of pannexin 1 channels in load-induced skeletal response. Annals of the New York Academy of Sciences, 1442(1), 79-90.

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Western Blot Protein Detection

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Protein samples and pre-stained molecular weight markers (Bio-Rad, Hercules, CA, USA) were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Medford, MA, USA). The membranes were incubated overnight at 4 °C in blocking solution containing specific antibodies (diluted at 1:1000). After being washed in PBS-Tween, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (diluted at 1:5000). The proteins recognized by the antibodies were visualized with an Immobilon western detection kit (Millipore).

Yang D., Okamura H., Morimoto H., Teramachi J, & Haneji T. (2016). Protein phosphatase 2A Cα regulates proliferation, migration, and metastasis of osteosarcoma cells. Laboratory investigation; a journal of technical methods and pathology, 96(10).

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Quantification of Protein Expression in Transfected Cells

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System (Thermofisher Scientific, UK). Cells were harvested in 2% Formaldehyde, and GFP expression was quantified via flow cytometry using the FACS caliber system (BD Biosciences, UK). Data was analysed using CellQuestPro software (BD Biosciences, UK).
Fluorescent intensity is reported at 4% gating to untreated cells.
For determination of expression of the HPV-16 E6 and E7 proteins, cells were harvested in radio immune precipitation assay (RIPA) buffer 48 h following transfection. Samples (20 μg) were electrophoresed through a 4-12% sodium dodecyl (SDS)-polyacrylamide gel (Life Technologies, UK), transferred onto a nitrocellulose membrane (Hybond-C, Amersham; UK) and probed with HPV-16 E6 and HVP-16 E7 antibodies (Abcam; UK). GAPDH was used as a loading control (Sigma; UK). Levels of protein expression were assessed using an Immobilon western detection kit (Millipore; UK). X-ray films were scanned using benchtop UV transilluminators (UVP Products Ltd) and density was calculated using Image J (http://rsbweb.nih.gov/ij/) incorporating correction of loading controls.

Cole G., Ali A.A., McCrudden C.M., McBride J.W., McCaffrey J., Robson T., Kett V.L., Dunne N.J., Donnelly R.F, & McCarthy H.O. (2018). DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V, 127.

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Oleocanthal Modulates Inflammatory Signaling in Chondrocytes

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Primary human OA chondrocytes were plated in 12-well plates, stimulated with oleocanthal 1-5 µM 12 hours and then incubated with LPS (250 ng/mL) during 24 hours or 30 minutes.
Proteins were extracted using a lysis buffer with a commercial protease inhibitor cocktail (Thermo Fisher). SDS-PAGE and blotting procedure were carried on as previously described [13] . Immunoblots were incubated with the appropriate antibody (iNOS, IkB, p-ERK-1/2 and NF-kB p65 from Cell Signaling MA, USA; COX-2 from DAKO, Denmark; MMP13 from Santa Cruz Biotechnology, USA; ERK1/2 and p-38 from Upstate, p-p38 from Millipore; Lamin B1 from Boster, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore Massachusetts, USA) using horseradish peroxidase-labelled secondary antibody. To confirm equal loading in each sample, the membranes were stripped in glycine buffer at pH 2 and re-blotted with an anti-GAPDH antibody (Sigma). The images were captured and analysed with an EC3 imaging system (UVP). Data obtained were further validated by densitometric analysis using Image J software.

Scotece M., Conde J., Abella V., López V., Francisco V., Ruiz C., Campos V., Lago F., Gomez R., Pino J, & Gualillo O. (2018). Oleocanthal Inhibits Catabolic and Inflammatory Mediators in LPS-Activated Human Primary Osteoarthritis (OA) Chondrocytes Through MAPKs/NF-κB Pathways. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 49(6).

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