After 48 h of transfection, the cells of each group were collected, and the total protein was extracted and quantified. After that, the same amount of protein sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). After the film was sealed with 5% skim milk at room temperature for 1 h, the sample was incubated overnight with primary antibody (1:5000) at 4 ℃, and then incubated with secondary antibody (1:3000) at room temperature for 2 h. The Enhanced Chemiluminescence (ECL) detection kit (Millipore, USA) was used for relevant detection. TBST was washed three times, each time for 10 min. super-Enhanced Chemiluminescence (ECL) detection kit (Millipore, USA) was used to develop the images. The images were captured by a Bio-Rad gel imaging system and repeated three times, and the results were analyzed by ImageJ software.
Enhanced chemiluminescence ecl detection kit
Manufactured by Merck Group
Sourced in United States
The Enhanced Chemiluminescence (ECL) detection kit is a laboratory tool used to detect and quantify proteins in Western blot analysis. The kit produces a luminescent signal when it reacts with the target protein, allowing for sensitive and accurate detection.
Automatically generated - may contain errors
Lab products found in correlation
Gel imaging system, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Las 4000, by Cytiva (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc propidium iodide apoptosis detection kit, by Keygen Biotech (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Ros assay kit, by Keygen Biotech (1 mentions)
Sp600125, by Beyotime (1 mentions)
Sb203580, by Beyotime (1 mentions)
Pd98059, by Beyotime (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
P stat1, by Cell Signaling Technology (1 mentions)
Jnk1 2, by Cell Signaling Technology (1 mentions)
P jnk1 2, by Cell Signaling Technology (1 mentions)
5 protocols using enhanced chemiluminescence ecl detection kit
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Protein Samples
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Cells, sEVs or mouse tissues were lysed in SDS-lysis or RIPA buffer. The protein samples were loaded into SDS–PAGE gels and then transferred onto 0.45-µm or 0.22-µm nitrocellulose membranes (Axygen). Membranes were blocked with 5% skimmed milk at room temperature for 1 h and incubated with primary antibodies at 4 °C overnight, following by incubation with the HRP-conjugated secondary antibodies at room temperature for 1 h. Finally, the membranes were visualized with an Enhanced Chemiluminescence (ECL) Detection kit (Millipore) and by using Image Quant LAS 4000 Mini (GE Healthcare Bio-Sciences AB). The primary antibodies used in the experiments are provided in Supplementary Table 2 .
3
Apoptosis Assay Protocol in DMEM
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Dulbecco's Modified Eagle Medium (DMEM), DMEM/F12, fetal bovine serum (FBS), phosphate buffer saline (PBS) and 0.25% trypsin were obtained from Gibco BRL, Life Technologies (Grand Island, NY, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), N-acetyl-L-cysteine (NAC), caspase-9 specific inhibitor (z-LEHD-fmk), Hoechst 33342 assay kit, annexin V-FITC/ propidium iodide (PI) apoptosis detection kit, mitochondrial membrane potential (MMP) assay kit, and ROS assay kit were purchased from KeyGen Biotech (Nanjing, China). SP600125, SB203580, and PD98059 were purchased from Beyotime (Nanjing, China). Primary antibodies against JNK, p-JNK, p38, p-p38, procaspase-3, Bcl-2 homologous antagonist/killer (Bak), B-cell lymphoma-xL (Bcl-xL), myeloid cell leukemia-1 (Mcl-1) and secondly antibodies were provided by Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cytochrome c (Cyt c), procaspase-9, extracellular signal-regulated kinase (ERK), p-ERK and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyvinylidene difluoride (PVDF) membranes and enhanced chemiluminescence (ECL) detection kit were purchased from Millipore (Billerica, MA, USA).
4
Western Blot Analysis of Immune Signaling
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Protein lysates extracted from different groups were separated via 8–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in Tris/glycine buffer (25 mM Tris and 250 mM glycine). The primary antibodies specific for TLR3 (115–130 kD), TLR4 (100–135 kD), TLR7 (140 kD), p-STAT1 (84, 91 kD), STAT1 (84, 91 kD), p-ERK1/2 (42, 44 kD), ERK1/2 (42, 44 kD), p-P38 (43 kD), P38 (43 kD), p-JNK1/2 (46, 54 kD), JNK1/2 (46, 54 kD), p-P65 (65 kD) and P65 (65 kD) were purchased from Cell Signaling Technology. HEV ORF3 (13.5 kD), p-IRF3 (47 kD), IRF3 (47 kD), p-IRF7 (54 kD) and IRF7 (54 kD) antibodies were acquired from Bioss (Beijing, China) and GFP (27 kD) from Wanleibio (Jilin, China). GAPDH (36 kD) antibody was purchased from Proteintech Group (Wuhan, China). Secondary antibodies were obtained from Pierce (1:5000 dilution). Bands were transferred to polyvinylidene fluoride (PVDF, Millipore, Billerica, MA) and proteins detected using the enhanced ECL chemiluminescence detection kit (Millipore, Billerica, MA) on a ChemiDoc XRS + imaging system (Bio-Rad, CA, USA).
5
Quantification of Proteins in HepG2.2.15 Cells
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The quantification of protein was performed in HepG2.2.15 cells using Western blot analysis, as previously reported [20 (link)]. Rabbit anti-ISG15, rabbit anti-pSTAT1, rabbit anti-STAT1, rabbit anti-pJAK1 and rabbit anti-JAK1 were purchased from cell signaling technology (CST, Danvers, MA), rabbit anti-MxA was purchased from Genetex (Irvine, USA), rabbit anti-IFIT1 was purchased from Bioss (Beijing, China), rabbit anti-USP18 and mouse monoclonal anti-HBcAg were purchased from Abcam (Cambridge, UK), rabbit anti-GAPDH was purchased from Protein Tech Group (Wuhan, China). Secondary antibodies were added for signal detection. An enhanced ECL chemiluminescence detection kit (Millipore, Billerica, MA) was used for detection. The quantification of protein bands related to GAPDH was performed by analysis with FUSION FX imaging system (Vilber, French).
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