The colon tissues were homogenized, and protein concentration was performed using the BCA Protein Assay Kit (NCM, Suzhou, China). Then, 25 µg proteins were separated with 12% SDS-PAGE gel and then transferred to PVDF membrane. The membranes were incubated with primary antibodies against toll-like receptor 4 (TLR4), P-p65, p65, P-p38, p38, p-ERK1/2, ERK1/2, P-JNK, JNK, or GAPDH (Affinity Biosciences, Jiangsu, China), followed by incubation with secondary antibodies. The signals were developed using an ECL Western blot detection kit (Affinity Biosciences, Jiangsu, China) and analyzed by Image J.
Ecl western blot detection kit
Manufactured by Affinity Biosciences
Sourced in China
The ECL Western blot detection kit is a laboratory instrument used to visualize and analyze protein samples. It utilizes a chemiluminescent detection method to enable the identification and quantification of specific proteins within a sample.
Automatically generated - may contain errors
3 protocols using ecl western blot detection kit
1
Toll-like Receptor Signaling Pathway Analysis
2
Quantifying Tumor Protein Levels
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We extracted proteins from normal and adjacent tumor tissues and tested their concentrations using BCA protein kits (Boster, Wuhan, China). Target proteins were isolated with SDS-PAGE (10%), and transferred to nitrate fiber membrane (4 ℃, 270 mA, 1.5 h). Then we sealed the nitrate fiber membrane with 5% skim milk powder for 3 h. The membrane was incubated with rabbitanti-CASP3 antibody (1:2,000; cat. no. ab184787; Abcam) or mouse anti‑glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:5,000; cat. no. ab8245; Abcam) orrabbit anti-TP53 antibody (1:500; cat. no. M00001-4; Boster) orrabbit anti-β-actin antibody (1:500; cat. no. WL0002d; Wanleibio) at 4 ℃ over-night. Wash unbound protein 3 times with 5% TBST solution., and the membrane was incubated with HRP Goat Anti-Rabbit IgG (H+L) secondary antibody (1:4,000; AS014; Abclonal) or HRP Goat Anti-Mouse IgG (H+L) secondary antibody (1:4,000; E-AB-1001; ELAbscience) for 1 h. Following washing with TBST (3 times). ECL western blot detection kit (Affinity Biosciences, USA) was used to visualize the protein bands. β-actin was used as internal references for protein. The molecular weight and net optical density of target band were analyzed by Image J. analysis software, and the results represented the relative content of target protein.
3
Protein Expression Analysis of Liver Tissue
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The liver tissues were homogenized, and protein concentration was performed using the BCA Protein Assay Kit (NCM, Suzhou, China). Then, 25 μg proteins were separated with 12% SDS-PAGE gel and then transferred to the PVDF membrane. The membranes were incubated with primary antibodies against Toll-like receptor 4 (TLR4), p65, phosphorylated (P)-p65, p38, P-p38, extracellular signal-regulated kinase (ERK1/2), p-ERK1/2, Jun N-terminal kinase (JNK), P-JNK (Affinity Biosciences, Changzhou, China), phosphatidylinositol 3-hydroxy kinase (PI3K), P-PI3K, protein kinase B (AKT), P-AKT, Foxo1, P-Foxo1, mammalian target of rapamycin (mTOR), P-mTOR, glycogen synthase kinase (GSK3β), P-GSK3β, LC3, Beclin1, Keap1, Nrf2, Heme Oxygenase-1 (HO-1), NOD-like receptor thermal protein domain associated protein 3 (NLRP3), caspase1 or GAPDH (Abcam, Cambridge, England), followed by incubation with secondary antibodies. The signals were developed using an ECL Western blot detection kit (Affinity Biosciences, Changzhou, China) and analyzed by Image J.
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