Differentiated cells were harvested and lysed in RIPA buffer supplemented with proteinase inhibitor cocktail (MedChemExpress). Proteins were separated on 10% SDS-PAGE gel and electro-transferred to PVDF membranes (Millipore). The membranes were blocked in blocking solution (5% skim milk in PBS) for 1 h at room temperature, and incubated overnight at 4°C with primary antibodies against JAK2 (1:1000, Cell Signaling Technology), p-JAK2 (Tyr1007/1008, 1:1000, Abcam), STAT3 (1:2000, Cell Signaling Technology), p-STAT3 (Tyr705, 1:2000, Abcam), HIF-1a (1:500, Santa Cruz Biotechnology), cyclin D1 (1:5000, proteintech), c-Myc (1:1000, Abmart), GAPDH (1:2000, Affinity) and β-actin (1:3000, Affinity). Thereafter, the membranes were incubated at room temperature for 1 h with horseradish peroxidase-linked secondary antibodies (1:2000, Wanleibio). The protein bands were detected by using an enhanced chemiluminescence kit (Wanleibio).
Enhanced chemiluminescence kit
Manufactured by Wanlei
Sourced in China
The Enhanced chemiluminescence kit is a laboratory product designed to detect and quantify proteins in Western blot analysis. The kit utilizes a chemiluminescent reaction to produce light, which is then detected and measured to determine the presence and abundance of specific proteins.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
C myc, by Abmart (1 mentions)
Proteinase inhibitor cocktail, by MedChemExpress (1 mentions)
Hif 1a, by Santa Cruz Biotechnology (1 mentions)
P jak2, by Abcam (1 mentions)
β actin, by Affinity Biosciences (1 mentions)
Gapdh, by Affinity Biosciences (1 mentions)
Cyclin d1, by Proteintech (1 mentions)
P stat3, by Abcam (1 mentions)
Anti trim21 antibody, by Abcam (1 mentions)
Sting d2p2f rabbit mab, by Cell Signaling Technology (1 mentions)
Irf 3 rabbit mab, by Cell Signaling Technology (1 mentions)
Gapdh antibody, by Wanlei (1 mentions)
Gapdh, by Proteintech (1 mentions)
5 protocols using enhanced chemiluminescence kit
1
Western Blot Analysis of JAK-STAT Pathway
2
NF-κB DNA Binding Assay
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NF-κB DNA-binding activity was detected using an NF-kB EMSA kit (cat no. BITF001; Viagene Biotech, Inc., Tampa, FL, USA), according to the manufacturer's protocol. Nuclear proteins were extracted and quantified as aforementioned. Proteins (25 µg) were diluted in 5 µl PBS and incubated with 0.5 µl biotin-labeled NF-κB specific probes (0.2 µmol/l; cat no. TF001BP; Viagene Biotech, Inc.) at room temperature for 20 min. The NF-κB-specific recognition sequence is: 5′-AGTTGAGGGGACTTTCCCAGGC-3′. The reaction mixtures (10 µl) were electrophoresed on 6.5% non-denaturing polyacrylamide gel at 180 V for 80 min. Protein-DNA complexes were electrically transferred onto nylon membranes, cross-linked under an ultraviolet lamp for 10 min and specific bands were detected by HRP-conjugated streptavidin and visualized using the Enhanced Chemiluminescence kit (Wanleibio).
3
Western Blot Analysis of Immune Signaling
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The total protein from mouse corneas (3 corneas/sample/group) and HCE cells was extracted and prepared in a standardized manner for Western blotting. After SDS-PAGE, transfer and blocking with 5% non-fat milk at room temperature for an hour on a shaker, membranes were incubated overnight at 4°C with anti-TRIM21 antibody (Abcam, United Kingdom, used at 1:1000), STING (D2P2F) Rabbit mAb (Cell Signaling Technology, China, 1:1000), IRF-3 Rabbit mAb (Cell Signaling Technology, 1:1000), phospho-IRF3 Rabbit mAb (Cell Signaling Technology, 1:1000) and GAPDH antibody (Wanlei, China, 1:1000) as primary antibodies. Followed by the membranes were washed three times in Tris-buffered saline. Then, the membranes incubated with goat horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Beyotime, China, 1:5000) as secondary reagents for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (Wanlei, China) was utilized to visualize the membrane. The densitometry analysis was performed using ImageJ 6.0 software.
4
Western Blot Protein Extraction and Analysis
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Total protein was extracted by sodium dodecyl sulfate lysis buffer (1% sodium dodecyl sulfate, 5% glycerol, 1 mM ethylene diamine tetraacetic acid (EDTA), 25 mM Tris, and 150 mM NaCl) with phenylmethylsulfonyl fluoride. Protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, United States). After blocking with 5% non-fat milk at room temperature for 1 h, membranes were probed with primary antibodies at 4°C overnight. Membranes were then washed using Tris-buffered saline with Tween 20 (TBST) for four times and incubated with corresponding secondary antibodies at room temperature for 1 h. Bound antibodies were visualized using an enhanced chemiluminescence kit (Wanleibio, Dalian, China). Primary antibodies were dissolved in TBST with 3% bovine serum albumin (1:1000). Secondary antibodies were dissolved in TBST (1:5000).
5
Protein Extraction and Western Blot Analysis
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Before protein extraction, cells were washed by 1 × PBS to remove the culture media. Cells were collected and lysed with BC-200 lysis buffer (20 mM HEPES buffer, pH 7.9, containing 200 mM KCl, 1 mM EDTA, 10 mM β-mercaptoethanol, 0.2 mM PMSF, 0.1% NP-40, and 10% glycerol). Protein samples were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk at room temperature for 1 h, the membranes were probed with primary antibodies at 4°C overnight. Then, after four times 1 × TBS with Tween-20 wash, we incubated the membranes with a corresponding secondary antibody at room temperature for 1 h. The primary antibodies used in this study included P53 and CDKN1A (Santa Cruz, Dallas, TX, USA), Caspase3 and cleaved-Capase3 (Abcam, Cambridge, UK), GAPDH (Proteintech, Wuhan, China), and the secondary antibodies includes goat anti-rabbit antibody and goat anti-mouse antibody. The secondary antibodies were obtained from Jackson ImmunoResearch Inc. (West Grove, PA, USA). Bound antibodies were visualized using an enhanced chemiluminescence kit (Wanleibio, Dalian, China)
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