Cells were lysed in RIPA lysis buffer (Fdbio science, Hangzhou, China) containing PMSF (Genstar) and then centrifuged at 4°C to collect the supernatant. Proteins were quantified using a BCA Protein Assay Kit (Beyotime, Shanghai, China), and equal samples were separated by 10% SDS‒PAGE and then transferred to PVDF membranes. After being blocked in 4% skimmed milk (Ruishu Biotechnology, Zhengzhou, China), the membranes were incubated with the primary antibody overnight at 4°C. The following primary antibodies were used: anti-MyoD (1:1000; Abcam, Cambridge, UK), anti-Myf5 (1:1000; Abcam), anti-MyoG (1:1000; Abcam), anti-MyHC (1:1000; Abcam), anti-Deltex2 (1:1000; Abcam), anti-Pax7 (1:1000; Abcam), anti-Ub (1:1000; Cell Signaling Technology, Beverly, USA), anti-Flag (1:1000; Cell Signaling Technology), anti-HA (1:1000; Cell Signaling Technology), anti-H3 (1:1000; Cell Signaling Technology), anti-GAPDH (1:5000; Bioworld) and β-tubulin (1:1000; Cell Signaling Technology). After three washes in TBS-Tween 0.1%, the membranes were incubated with the corresponding HRP-conjugated secondary antibody (Cell Signaling Technology) at room temperature for 1 h. Immunoblots were detected using ECL (FDbio Science) and a BioLight system (Guangzhou, China).
Enhanced chemi luminescence (ecl)
Manufactured by Fdbio
Sourced in China
The ECL (Enhanced Chemiluminescence) is a laboratory equipment used for the detection and quantification of proteins in Western blotting experiments. It utilizes a chemiluminescent substrate to produce a light signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti h3, by Cell Signaling Technology (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Fdbio (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by GenStar (1 mentions)
Anti myod, by Abcam (1 mentions)
Anti myf5, by Abcam (1 mentions)
Anti myog, by Abcam (1 mentions)
Anti deltex2, by Abcam (1 mentions)
Anti pax7, by Abcam (1 mentions)
Anti ub, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Bioworld Technology (1 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Anti myhc, by Abcam (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
5 protocols using enhanced chemi luminescence (ecl)
1
Protein Expression Analysis in Myogenesis
2
Western Blot Analysis of Cellular Proteins
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Cells were lysed with 1X sodium dodecyl sulfate lysis buffer. Total protein was quantified, separated by SDS‒PAGE, and transferred onto PVDF membranes (Millipore, MA, USA). The target proteins were probed with antibodies against GPx2 (#ab140130, Abcam), KYNU (#11796-1-AP, Proteintech), AhR (#67785-1-Ig, Proteintech), E-cadherin (#3195 S, Cell Signaling Technology), N-cadherin (#13116 S, Cell Signaling Technology), Vimentin (#5741 S, Cell Signaling Technology) and GAPDH (#60004-1-Ig, ProteinTech). Anti-mouse IgG (926-6807, Invitrogen) and anti-rabbit IgG (926-68070, Invitrogen) were used as secondary antibodies. Finally, the protein bands were visualized with enhanced chemiluminescence (ECL; Fdbio Science, Hangzhou, China). Intensity was measured by ImageJ software.
3
Western Blot Analysis of Signaling Proteins
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Protein lysates derived from BC cells were separated by 12% SDS-PAGE gels and then transferred to 0.45-μm PVDF membranes. After being blocked with 10% skim milk for 30 minutes at room temperature, the membranes were incubated with antibodies against hnRNP-F, AKT, p-AKT, PI3K, p-PI3K, FOXO1, p-FOXO1 (all 1:1000) and GAPDH (1:5000) at 4 °C overnight, followed by an incubation with appropriate HRP-conjugated secondary antibodies (1:5000). Immunoreactivity was visualized by ECL (Fdbio Science, Hangzhou, China) and an enhanced chemiluminescence (ECL, Pierce, Rockford, IL, USA) detection system for blot visualization. The protein levels were normalized to those of GAPDH.
4
Western Blot Analysis of Angiogenic Factors
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Total cellular proteins were extracted using RIPA buffer. Protein concentrations were determined using the BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the membranes were blocked in 5% skimmed milk in TBST at room temperature for 1 h. The blots were incubated at 4° C overnight with the following primary antibodies (Abcam, Cambridge, MA, USA): anti-VEGF (1:1000), anti-angiogenin-1 (1:1000), anti-MMP9 (1:1000), anti-MMP14 (1:1000), anti-p-Akt (1:1000), anti-Akt (1:1000), anti-p-ERK (1:1000), anti-ERK (1:1000), anti-VEGFC (1:1000), anti-β-actin (1:1000). Then, the blots were incubated with HRP-conjugated secondary antibodies (1: 5000) for 50 min at room temperature. Subsequently, blots were developed using ECL (Fdbio Science, Hangzhou, China).
5
Immunoblotting of Cell Lysates
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Total cell lysates and immunoprecipitates were separated by sodium dodecyl sulfate (SDS)-12% polyacrylamide gel electrophoresis (PAGE) and blotted onto to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The following primary antibodies were used for immunodetection: AnxA2, ILK, and p-p38 (Santa Cruz Biotechnology) at 1:1000- to 1:2000-fold dilutions. After incubation overnight at 4 °C, blots were exposed to the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (Huabio, Hangzhou, China). Immunoreactive bands were visualized by electrochemiluminescent (ECL) (FDbio Science, Hangzhou, China). β-actin served as loading controls.
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