ZF4 cells were lysed in RIPA buffer with 1% PMSF (ComWin Biotech, China). Zebrafish embryos were manually de-yolked and lysed in the lysis buffer. Equal amount of protein samples were separated with 10% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membrane was blocked for 1 h in TBST containing 5% milk at room temperature, followed by 1 h incubation with specific primary antibodies against zebrafish Cdc25a (1:500) (Homemade polyclonal antibody prepared using prokaryotic expression system, affinity-purified and generated in rabbits), Cdk2 (Cyclin-dependent kinase 2, 1:1000) (Aviva Systems Biology, USA), Hif-1α (1:2000) (Homemade polyclonal antibody generated in rabbits) or β-actin (1:500) (Boster, China). The blot was detected with IRDye 800CW anti-rabbit secondary antibody (1:10,000) (Li-Cor Biosciences, USA) and visualized using Odyssey CLx Infrared Imaging System (Li-Cor Biosciences).
Hif 1α
Manufactured by Boster Bio
Sourced in China
Hif-1α is a protein that plays a critical role in the cellular response to low oxygen levels. It functions as a transcription factor, regulating the expression of genes involved in various physiological and pathological processes related to hypoxia.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
β actin, by Boster Bio (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Irdye 800cw anti rabbit secondary antibody, by LI COR (1 mentions)
Hsp70, by Boster Bio (1 mentions)
Odyssey fc machine, by LI COR (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Radioimmunoprecipitation assay (ripa), by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Protease inhibitor, by Boster Bio (1 mentions)
Gapdh, by Proteintech (1 mentions)
4 protocols using hif 1α
1
Zebrafish Protein Expression Analysis
2
Quantification of Hif-1α and Hsp70 in M. amblycephala
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Total proteins from M. amblycephala liver, spleen, brain, gill, and kidney (n = 5 for each group) were quantified and separated on 8% SDS-PAGE, then transferred to nitrocellulose membranes. The membranes were incubated for 2 h with polyclonal antibodies, Hif-1α (1:500) and Hsp70 (1:200) (Boster, China), respectively, and then anti-rabbit IRDye 800CW-labeled secondary antibody (1:10,000) at room temperature for 1 h, and observed using Odyssey Fc machine (Licor Biosciences, USA). Gray values of every band were further calibrated and measured by ImageJ 1.46r (NIH, USA).
3
Western Blot Analysis of Cellular Signaling Proteins
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Total protein was extracted using RIPA (sigma, Germany) lysis buffer containing protease inhibitor PMSF (Invitrogen, United States). Protein concentration was detected by BCA protein assay kit (Invitrogen, United States). Total of 20 µg protein was electrophoresed in 10% SDS-PAGE gels and transferred onto PVDF membranes (Merk, Germany). The membranes were blocked with TBST solution containing 5% milk for 2.5 h at room temperature, then probed with the primary antibody overnight and the secondary antibody for 1 h. Wash the membranes with TBST solution three times for 5 min each before and after incubation of secondary antibodies. Finally, the blots were captured by Amersham imager 600 (GE, United States) and the quantitative results were analyzed by ImageJ analysis software. The list of antibodies is as follows: Ferritin (Huabio, China), Hif1α (Boster, China), LC3 (Cell Signaling Technology, United States), P62 (Cell Signaling Technology, United States), Actin (Huabio, China) and the secondary antibody (Huabio, China).
4
Western Blot Analysis of BMSC Proteins
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Proteins were lysed from BMSCs in a 6-well plate using a RIPA lysis buffer containing protease inhibitors (Boster Biological Technology co.ltd, China). After measurement of the protein concentration with BCA protein assay kit (Boster Biological Technology co.ltd, China), proteins of the same quality (30 μg) were separated on sodium dodecyl sulfate-polyacrylamide electrophoresis gels and transferred to 0.45 μm PVDF membranes (Millipore). The membranes were blocked with 5% milk resolved in Tris buffered saline-Tween buffer, and probed by diluted antibodies. Primary antibodies used included the following: Hif1α (Boster Biological Technology co.ltd, China), P16 (Abcam, UK), p21 (Boster Biological Technology co.ltd, China), p53 (Cell Signaling Technology, USA), and GAPDH (Proteintech Group, Inc., USA). The grayscale of the band were quantified using ImageJ software. The relative value of the target gene expression was obtained by dividing the gray value of the target gene by the gray value of the internal reference band (GAPDH).
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