Liver tissues were collected and homogenized in RIPA Lysis Buffer (Cell Signaling Technology, USA) containing PMSF and centrifuged at 12,000g at 4°C for 15 min. The concentration of protein samples was measured by BCA Protein Assay (Cell Signaling Technology, USA). A total of 35 µg protein for each sample were loaded, diluted with 5 × SDS‐PAGE Sample Loading Buffer (Beyotime, Shanghai, China), and heated at 95°C for 5 min. Then, the proteins underwent electrophoresis by 10% SDS‐PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Then, the membranes were incubated in blocking buffer for 1.5 hr. Afterward, the membranes were incubated with the primary antibodies of TLR4 (1:1,000, ProteinTech, USA), MyD88, NF‐κB p65, IκBα, and p‐IκBα (1:1,000, Cell Signaling Technology, USA), β‐actin (1:10,000, ProteinTech, USA), overnight at 4°C, washed with TBST (tris buffered saline with Tween), and incubated with secondary antibody (1:8,000, ProteinTech, USA) for 2 hr at room temperature. After five 10‐min washes, the expressions of proteins were analyzed by ECL (ProteinTech, USA) detection. The gray value of each band was measured using Image J software.
Enhanced chemi luminescence (ecl)
Manufactured by Proteintech
Sourced in China, United States
ECL is a chemiluminescent detection reagent used in Western blotting to visualize proteins. It generates a luminescent signal when exposed to the enzyme-labeled secondary antibody, which can be detected using photographic film or a digital imaging system.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidence fluoride membranes, by Merck Group (2 mentions)
Myd88, by Cell Signaling Technology (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
β actin, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
5 protocols using enhanced chemi luminescence (ecl)
1
Western Blot Analysis of Liver Proteins
2
Protein Expression Analysis Protocol
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Equal amounts of protein obtained from cells or liver tissue samples were run in vertical electrophoresis unit, then transferred to the PVDF membrane (Millipore, USA), and blocked with 5% nonfat milk in TBS-T (TBS + 0.4% Tween-20) for 2 h. Subsequently, the PVDF membrane was incubated with corresponding primary antibodies at a dilution of 1:1000 except GAPDH and β-actin at 4 °C overnight, followed by three washes with TBS-T. HRP-conjugated secondary antibodies were, respectively, used to combine primary antibodies for 2 h, followed by three washes in TBS-T for 8 min each. Finally, blots were incubated with ECL (Proteintech, USA). The intensity of bands was quantified using software Image J.
3
Protein Analysis by Western Blot
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Cell lysates and exosomes including purified membrane vesicles were separated by SDS‐PAGE and were then transferred to polyvinylidence fluoride membranes (Millipore, Darmstadt, Germany). The membrane was sealed with 5% non‐fat milk for more than 1 h at room temperature and incubated with the desired primary antibodies overnight at 4 °C. Post incubation with HRP‐conjugated secondary antibodies was performed for 1 h at room temperature, then detection was carried out using enhanced chemiluminescence reagent (ECL) (Protein Tech, China).
4
Immunoblotting Analysis of Cell and Extracellular Vesicle Proteins
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Cell and sEV lysates were separated using the SDS-PAGE and were then transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Then the membrane was sealed with 5% non-fat milk for 1.5 h at room temperature and incubated with the desired primary antibodies (CD63, CD9, Alix, TSG101, STAT6, LYN, and β-actin) overnight at 4°C. Post-incubation with HRP-conjugated secondary antibodies was performed for 1.5 h at room temperature, and then detection was carried out using an enhanced chemiluminescence reagent (ECL) (Protein Tech, China).
5
Protein Immunoblotting of Cell Lysates
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Cell lysates and exosomes including purified membrane vesicles were separated by SDS-PAGE and were then transferred to polyvinylidence fluoride membranes (Millipore, Darmstadt, Germany). The membrane was sealed with 5% non-fat milk for more than 1 h at room temperature and incubated with the desired primary antibodies overnight at 4 °C. Post incubation with HRP-conjugated secondary antibodies was performed for 1 h at room temperature, then detection was carried out using enhanced chemiluminescence reagent (ECL) (Protein Tech, China).
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