Ecl chemiluminescent method

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The ECL chemiluminescent method is a laboratory technique used to detect and quantify specific proteins or molecules in a sample. It relies on the emission of light generated by a chemical reaction to produce a measurable signal, which can be used to identify and analyze the target analyte.

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ECL method (19 citations)

4 protocols using ecl chemiluminescent method

Protein Expression Analysis of Lung Tissues

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Lung tissues or cells were lysed with RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride. The supernatants were collected by centrifuged and total protein concentration was qualified using a BCA Protein Kit (Beyotime Biotechnology, Shanghai, China). Then equal quantities of protein samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes (Meckmillipore, Germany) that were consequently blocked with 5% fat-free milk for 1 hr. After that, the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies were used as follows: anti-HMGB1 (1:1000; Cat#6893),anti-TLR4 (1:1000; Cat#14358), anti-NF-κB (1:1000; Cat#8242), anti-p-NF-κB (1:1000; Cat#3033), cleaved caspase-3 (1:1000; Cat#9661) (Cell Signaling Technology); anti-Bcl-2 (1:1000; Cat#ab182858), anti-Bax (1:1000; Cat#ab32503), and anti-β-actin (1:1000; Cat#ab8227) (Abcam). Following extensively rinsing in TBST, the membranes were incubated with HRP-conjugated secondary antibodies and further detected using ECL chemiluminescent method (Millipore, Billerica, MA, USA). The blots were quantified with ImageJ.

Liu M., Lu J., Zhang Q., Zhang Y, & Guo Z. (2021). Clara cell 16 KDa protein mitigates house dust mite-induced airway inflammation and damage via regulating airway epithelial cell apoptosis in a manner dependent on HMGB1-mediated signaling inhibition. Molecular Medicine, 27, 11.

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Western Blot Analysis of Lung Cell Proteins

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Lung tissues or cells were lysed with RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl uoride. The supernatants were collected by centrifuged and total protein concentration was quali ed using a BCA Protein Kit (Beyotime Biotechnology, Shanghai, China). Then equal quantities of protein samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes(Meckmillipore,Germany) that were consequently blocked with 5% fat-free milk for 1hr. After that, the membranes were incubated with primary antibodies at 4℃ overnight. The primary antibodies were used as follows: anti-HMGB1 (1:1000; Cat#6893),anti-TLR4 (1:1000; Cat#14358), anti-NF-κB (1:1000; Cat#8242), anti-p-NF-κB (1:1000; Cat#3033), cleaved caspase-3 (1:1000; Cat#9661) (Cell Signaling Technology); anti-Bcl-2 (1:1000; Cat#ab182858),anti-Bax (1:1000; Cat#ab32503), and anti-βactin (1:1000; Cat#ab8227) (Abcam).Following extensively rinsing in TBST, the membranes were incubated with HRP-conjugated secondary antibodies and further detected using ECL chemiluminescent method (Millipore, Billerica, MA, USA). The blots were quanti ed with ImageJ.

Liu M., Lu J., Zhang Q., Zhang Y., & Guo Z. (2021). Clara cell 16KDa protein mitigates house dust mite-induced airway inflammation and damage via regulating airway epithelial cell apoptosis in a manner dependent on HMGB1-mediated signaling inhibition.

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Western Blot Analysis of BEAS-2B Cells

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BEAS-2B cells were lysed with RIPA buffer containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride. The supernatants were collected by centrifuged and total protein concentration was qualified using a BCA Protein Kit(Beyotime Biotechnology, Shanghai, China). Then equal quantities of protein samples were separated by 8% SDS-PAGE and transferred onto PVDF membranes(Meckmillipore,Germany) that were consequently blocked with 5% fat-free milk for 1hr. After that, the membranes were incubated with primary antibodies at 4 overnight. The primary antibodies were used as follows: anti-HMGB1(1:1000),anti-TLR4(1:1000),anti-NF-κB(1:1000), anti-p-NF-κB(1:1000), anti-Bcl-2(1:1000),anti-Bax(1:1000), and anti-βactin(1:1000)(Cell Signaling Technology, Danvers, MA,USA).Following extensively rinsing in TBST, the membranes were incubated with HRP-conjugated secondary antibodies and further detected using ECL chemiluminescent method (Millipore, Billerica, MA, USA). The blots were quantified with ImageJ.

Liu M., Lu J., Zhang Q., Zhang Y., & Guo Z. (2020). Clara Cell 16KDa Protein Mitigates House Dust Mite-Induced Airway Inflammation and Damage via Regulating Airway Epithelial Cell Apoptosis in a Manner Dependent on HMGB1-Mediated Signaling Inhibition.

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Western Blot Analysis of Lung Cell Proteins

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