western blot original images
Description
western blot to compare differnces in key protein expression before and after hypoxia
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Steps to reproduce
Western blotting According to the manufacturer’s instructions, RIPA lysate (PC101, Shanghai Yase Biomedicine Technology Co., LTD.) was used to extract protein according to the standard protocol, and the protein concentration was determined by the BCA protein assay (ZJ102, Shanghai Yase Biomedicine Technology Co., LTD.). A sample of 30 μg was electrophoreted onto a 10% SDS-PAGE gel and then transferred to a nitrocellulose membrane (IPVH00010, Millipore). At room temperature, the membrane was blocked in TBST with 5% skim milk for 90 minutes. Then HSP90 (1:1000, 60318-1-Ig, Mouse, Proteintech), HRAS(1:500, AF02931, Rabbit, Hunan Aifang Biotechnology Co., LTD.), AKT1 (1:500, AF0016, Rabbit, Affinity), PIK3CA (1:500, AF4669, Rabbit, Affinity), VEGFA (1:500, AF5131, Rabbit, Affinity), HIF1A (1:1000, ab179483, Rabbit, Abcam), EDN1 (1:500, DF6125, Rabbit, Affinity), EDNRA (1:500, DF4923, Rabbit, Affinity), EDNRB (1:500, DF7104, Rabbit, Affinity), β-actin (1:500, 81115-1-RR, Rabbit, Proteintech). The primary antibodies were incubated at 4℃ for overnight. Subsequently, suitable secondary antibodies were incubated at room temperature for 1 hour. Bands were detected using an ECL luminescence kit (SQ101, Shanghai Yase Biomedical Technology Co., LTD.). Statistical analysis was performed by ImageJ software. The relative protein level for each sample was reflected by the gray value of the individual protein divided by the gray value of the respective β-actin.
Institutions
- University of Shanghai for Science and Technology