RIPA lysis buffer (KeyGene) was used for extracting total proteins. After measurement of protein concentration, proteins were subjected to SDS-PAGE, followed by transferring onto the PVDF membranes (Millipore, Billerica, MA, USA). After blockage using 5% skim milk (Beyotime, Shanghai, China), these membranes were immunoblotted with primary antibodies at 4°C for 10–14 h. Lastly, the combined signals were detected by an enhanced chemiluminescence kit (KeyGene) after incubation of corresponding secondary antibody. The antibodies including proliferating cell nuclear antigen (PCNA; 1:500, ab18197), Bcl-2 (1:500, ab196495), Bax (1:500, ab32503), Cleaved Caspase 3 (1:500, ab32042), IGFBP3 (1:1000, ab193910), β-actin (1:5000, ab227387), CD63 (1:1000, ab118307), TSG101 (1:500, ab30871), and HRP-conjugated IgG anti-rabbit (1:4000, ab205718) were commercially acquired from Abcam (Cambridge, MA, USA). All experiments were repeated three times.
Enhanced chemiluminescence kit
Manufactured by Keygene
Sourced in China
The Enhanced Chemiluminescence Kit is a laboratory product designed to detect and quantify protein expression levels. It utilizes a chemiluminescent substrate to generate a light signal proportional to the amount of target protein present in a sample. The kit provides the necessary reagents and materials to perform this analysis.
Automatically generated - may contain errors
Lab products found in correlation
Skim milk, by Beyotime (2 mentions)
Ab205718, by Abcam (2 mentions)
Ripa lysis buffer, by Keygene (2 mentions)
Ab32042, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab30871, by Abcam (1 mentions)
Ab18197, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab118307, by Abcam (1 mentions)
Ab227387, by Abcam (1 mentions)
Hrp conjugated anti rabbit igg, by Abcam (1 mentions)
Ab193910, by Abcam (1 mentions)
Tsg101, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
β actin, by Proteintech (1 mentions)
E cadherin e cad, by Proteintech (1 mentions)
N cadherin n cad, by Proteintech (1 mentions)
2 protocols using enhanced chemiluminescence kit
1
Protein Extraction and Immunoblotting Protocol
2
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total protein was extracted by lysing cells in RIPA lysis buffer (KeyGene, Nanjing, China). After measurement of protein concentration, equal amounts of protein (30 μg per lane) were subjected to 10 % SDS-PAGE. After transferring onto the PVDF membranes (Bio-Rad), the membranes were blocked in 5 % skim milk (Beyotime), followed by immunoblotting with primary antibodies at 4℃for 10–14 h. Next, the corresponding secondary antibody (ab205718; 1:4000; Abcam, Cambridge, UK) was applied in combination with primary antibodies. Lastly, the combined signals were visualized with an enhanced chemiluminescence kit (KeyGene). In this study, the primary antibodies were purchased from Proteintech (Rosemont, IL, USA): E-cadherin (E-cad; 20874–1-AP; 2000), N-cadherin (N-cad; 22018–1-AP; 1:2000), GLUT1 (21829–1-AP; 1:2000), LDHA (21799–1-AP; 1:5000), ARF6 (20225–1-AP; 1:500) and β-actin (20536–1-AP; 1:2000).
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