HIF-1α DNA binding activity was measured using a commercial assay kit (Abcam, ab133104). Briefly, nucleoprotein was extracted from HepG2 cells using a Nuclear Extraction Kit (Beyotime, China). Then, the obtained samples were added to the wells of transcription factor HIF-1α plate and incubated overnight at 4 °C. After incubation, diluted HIF-1α primary antibody was added to each well and incubated for 60 min at room temperature. Subsequently, diluted goat anti-rabbit HRP conjugate was added to each well and incubated for 60 min at room temperature. HIF-1α DNA binding activity were measured at 450 nm using a microplate reader (Biotek, USA).
Ab133104
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab133104 is a recombinant monoclonal antibody. It is designed to detect the protein target of interest in biological samples.
Automatically generated - may contain errors
6 protocols using ab133104
1
Quantifying HIF-1α DNA Binding Activity
2
HIF-1α Transcriptional Factor Assay
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Protein activity of HIF‐1α was measured using a commercially available HIF‐1α transcriptional factor assay kit (ab133104, abcam) according to the manufacturer's instructions.
3
Fractionation and Transcription Factor Analyses of BMDM and Muscle Tissue
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Nuclear and cytoplasmic fractionation of BMDM or muscle tissue lysates was carried out according to manufacturer instructions (Abcam, ab113474). Briefly, cells were collected with cell scraper and pelleted by centrifugation. Pellets were re-suspended in the pre-extraction buffer. After a centrifugation, supernatant containing cytoplasmic fraction was frozen down and stored. The pellet was again centrifuged and re-suspended in nuclear extraction buffer to generate the nuclear fraction. The nuclear fraction was used for HIF-1α, STAT3, and NF-κB p65 transcription assays according to the manufacturer’s instructions (Abcam; ab133104, ab207229, and ab133112, respectively).
4
HIF-1α Transcriptional Activity Assay
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Rat aortic endothelial cells (2 × 105) were stably transfected with HIF-1α luciferase reporter (RC0017, Panomics, Fremont, CA, USA) and seeded in complete media (DMEM with 10% FBS) in 24-well plates. Luciferase activity was measured on hour 24 with HIF-1 alpha Transcription Factor Assay (ab133104; Abcam, Cambridge, MA, USA) according to protocol. In brief, cells were lysed by each well being added with cell lysis solution. Then, lysates mixed with ATP mix served as the substrate for the chemiluminescent oxidation-reduction reaction which leads to light emission and oxy-luciferin generation. The reaction liquid was measured with a luminometer at a light of 560 nm.
5
HIF-1α Activity Quantification Assay
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Briefly, a specific double stranded DNA sequence containing the HIF-1α response element (5’-ACGTG-3’) is immobilized to the wells of a 96-well plate. The nuclear extract lysates from sh-Ctrl and sh-MIR210HG MDA-MB-231 and HCC1937 cells were harvested using the Nuclear Extraction kit (ab113474; Abcam, Cambridge, UK). Then, HIF-1α activity was detected by addition of a specific primary antibody directed against HIF-1α according to the manufacturer’s instruction (ab133104; Abcam, Cambridge, UK). A secondary antibody conjugated to HRP is added to provide a sensitive colorimetric readout at 450 nm.
6
Fractionation and Transcription Factor Analyses of BMDM and Muscle Tissue
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