Immobilon ecl system

Manufactured by Merck Group

Sourced in United States

The Immobilon ECL system is a detection system for chemiluminescent Western blotting. It generates a luminescent signal when exposed to peroxidase-conjugated secondary antibodies, allowing for the visualization and quantification of target proteins.

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Lab products found in correlation

Pvdf membrane, by Merck Group (3 mentions) Bca protein assay, by Beyotime (2 mentions) Ab8245, by Abcam (2 mentions) Anti egfp, by Takara Bio (1 mentions) Anti actin 1 19, by Santa Cruz Biotechnology (1 mentions) Anti flag m2 antibody, by Merck Group (1 mentions) Ab32575, by Abcam (1 mentions) Ab138492, by Abcam (1 mentions) Mmp12, by Proteintech (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Phospho stat3, by Cell Signaling Technology (1 mentions) Mmp 9, by Abcam (1 mentions) Ripa buffer, by Keygen Biotech (1 mentions) Gapdh, by Proteintech (1 mentions) Ab65080, by Abcam (1 mentions) Ab86707, by Abcam (1 mentions) Ab38898, by Abcam (1 mentions)

4 protocols using immobilon ecl system

SDS-PAGE Protein Analysis and Immunoblotting

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All of the protein extracts were heated at 99°C for 5 minutes in SDS-PAGE solubilising buffer (58 mmol/L Tris HCl, 10% glycerol, 2% SDS, 0.004% bromophenol blue, pH 6.8) containing 7.5% dithiothreitol. The proteins were separated by means of SDS-PAGE electrophoresis on a 10% polyacrylamide gel, and transferred to a PVDF membrane. After blocking, the membrane was incubated with anti-ICln [5] (link), anti-actin I-19 (Santa Cruz Biotechnology, Dallas, Texas, USA), anti 4.1R C-16 (Santa Cruz Biotechnology) or anti-4.1R EPB41 (Sigma-Aldrich), anti-EGFP (Clontech), monoclonal anti-GAPDH (clone GAPDH-71.1, Sigma-Aldrich), anti-pan cadherin ABT35 (Abcam plc, Cambridge, UK), or anti-FLAG M2 antibody (Sigma-Aldrich), diluted in the blocking buffer at 4°C overnight, followed by several washes, and then by the secondary HRP-conjugated antibody. The Immobilon ECL system (Millipore S.p.A., Vimodrone, Italy) was used for detection.
The PVDF membrane was always stained using the amido black staining procedure in order to assess the efficiency of protein transfer and verify equal loading.
The bands were densitometrically analysed using the ImageJ software.

Bazzini C., Benedetti L., Civello D., Zanoni C., Rossetti V., Marchesi D., Garavaglia M.L., Paulmichl M., Francolini M., Meyer G, & Rodighiero S. (2014). ICln: A New Regulator of Non-Erythroid 4.1R Localisation and Function. PLoS ONE, 9(10), e108826.

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Protein Expression Analysis in Cell Lysates

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Total lysates were prepared according to the manufacturer's recommendations (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were measured with the BCA protein assay according to the manufacturer's manual (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (30 μg) of protein were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). Membranes were incubated overnight at 4°C with mouse anti‐GAPDH antibody (ab8245, Abcam), mouse anti‐β‐actin antibody (66009‐1‐Ig, Proteintech), rabbit anti‐E‐cadherin antibody (#3195, Cell Signaling Technology), rabbit anti‐alpha smooth muscle actin antibody (α‐SMA) (ab32575, Abcam) and rabbit anti‐collagen I antibody (ab138492, Abcam). After several washing steps, the membrane was incubated with secondary horseradish peroxidase (HRP)‐conjugated antibody at room temperature for one hour. Detection was performed with the Immobilon ECL system (Millipore, S.p.A., Italy). Three independent experiments were carried out. The densitometric analyses of the bands were performed with ImageJ software.

Di T., Yang Y., Fu C., Zhang Z., Qin C., Sai X., Liu J., Hu C., Zheng M., Wu Y, & Bian T. (2020). Let‐7 mediated airway remodelling in chronic obstructive pulmonary disease via the regulation of IL‐6. European Journal of Clinical Investigation, 51(4), e13425.

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Protein Expression Analysis via Western Blot

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Total protein was extracted from cells using RIPA buffer (KeyGEN, China) supplemented with a protease inhibitor cocktail (CWBIO CW2200, China) and phosphatase inhibitor (CWBIO CW2383, China). Equal amounts (20 μg-40 µg) of protein were separated via SDS‒PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). After being blocked for 1 h in 5% milk, the membranes were incubated with antibodies targeting GAPDH (Proteintech, China), MMP9 (Abcam), MMP12 (Proteintech), STAT3 (Cell Signaling Technology), and phospho-STAT3 (Cell Signaling Technology) at 4°C overnight. After being incubated with secondary antibodies (Jackson ImmunoResearch, USA) for 1 h at room temperature, the protein bands were measured with the Immobilon ECL system (Millipore, S.p.A., Italy). ImageJ software was used for densitometric analyses.

Liu T., Zhang Z., Shen W., Wu Y, & Bian T. (2023). MicroRNA Let-7 Induces M2 Macrophage Polarization in COPD Emphysema Through the IL-6/STAT3 Pathway. International Journal of Chronic Obstructive Pulmonary Disease, 18, 575-591.

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Western Blot Quantification of Protein Targets

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Total lysates were prepared according to the manufacturer’s recommendations (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were measured with the BCA protein assay according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts (20 μg-40 µg) of protein were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were incubated overnight at 4°C with mouse anti-GAPDH (ab8245, Abcam), mouse anti-β-actin (66009-1-Ig, Proteintech), rabbit anti-NCOA4 (ab86707, Abcam), rabbit anti-ferritin (ab65080, Abcam), rabbit anti-Ptgs2 (#12282, CST), rabbit anti-MMP9 (ab38898, Abcam) and rabbit anti-MMP12 (22989-1-AP, Proteintech) antibodies. After several washing steps, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for one hour. Detection was performed with the Immobilon ECL system (Millipore, S.p.A., Italy). Three independent experiments were carried out. Densitometric analyses of the bands were performed with ImageJ software.

Liu J., Zhang Z., Yang Y., Di T., Wu Y, & Bian T. (2022). NCOA4-Mediated Ferroptosis in Bronchial Epithelial Cells Promotes Macrophage M2 Polarization in COPD Emphysema. International Journal of Chronic Obstructive Pulmonary Disease, 17, 667-681.

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