HUVEC or aortic tissue proteins were prepared using the RIPA Lysis buffer for immunoprecipitation containing Protease/Phosphatase Inhibitors Cocktail. According to the immunoprecipitation protocol, 0.5–1.0 μg of anti-p53 antibody (Proteintech, catalog 60283-2-Ig) was added to 0.5–1.0 ml of lysate sample prepared from 106 to 107 cells typically and the anti-p53 antibody-antigen reactions were completed at 4°C overnight on a rocking platform. Then 40 μl of Protein A+G Agarose (Beyotime) was added to the antibody-antigen lysate mentioned above, and gently incubated on a rocking platform for 1 h at 4°C. The Protein A+G Agarose was collected by centrifugation at 1,000 g for 5 min at 4°C, and the immune complexes in PBS were washed to remove nonspecific binding. The immune complexes were washed five times, and the final wash was removed as completely as possible. In the end, the samples were added the SDS loading buffer and heated at 100°C for 5 min to release both noncovalently bound anti-p53 antibodies and antibody fragments along with p53. Then the levels of p53 and p53 SUMOylation were detected by western blot immediately.
Anti p53 antibody
Manufactured by Proteintech
Sourced in China, United States
The Anti-p53 antibody is a laboratory reagent designed for the detection and analysis of the p53 protein, a key regulator of cell growth and division. This antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and immunoprecipitation, to identify and quantify the p53 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti caspase 3 antibody, by Proteintech (2 mentions)
Protein a g agarose, by Beyotime (1 mentions)
Chip kit, by Beyotime (1 mentions)
Rpmi 1640 medium, by Sartorius (1 mentions)
Anti gapdh antibody, by Abcam (1 mentions)
Anti γh2ax antibody, by Abcam (1 mentions)
Dmem medium, by Sartorius (1 mentions)
Anti p21 antibody, by Proteintech (1 mentions)
Anti β actin antibody, by Proteintech (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Anti caspase9 antibody, by Proteintech (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti gapdh antibody, by Proteintech (1 mentions)
Anti timp1 antibody, by Santa Cruz Biotechnology (1 mentions)
Anti α sma antibody, by Proteintech (1 mentions)
Ecl assay kit, by Beyotime (1 mentions)
Goat anti mouse igg h l hrp, by Jackson ImmunoResearch (1 mentions)
5 protocols using anti p53 antibody
1
Immunoprecipitation of p53 and Detection
2
ChIP-qPCR Analysis of p53 Binding
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ChIP assays were performed with a ChIP kit (Beyotime Biotechnology), and then the TIANamp Genomic DNA kit was used for DNA purification. Briefly, the cells were seeded in 10 cm culture plates at 5 × 105 cells (HT29), cultured in normal medium overnight, and then left in specific treatment at the 48 or 24 h time‐point before the subsequent test. The cells were harvested after fixation with formaldehyde and then quantified into 1 × 106 cells per pipe for ChIP assays. qPCR analysis was performed to detect DNA fragments immunoprecipitated with anti‐p53 antibody (1:200, Proteintech, 10442–1‐AP).
3
Phenylquinoline Derivatives Synthesis
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The compound library containing
120 small molecules was from the laboratory of Zhenyu Li. The phenylquinoline
derivatives were synthesized through the Suzuki coupling reaction
as in our previous description.33 (link) PFTα
(Cat#: T2707) was from Targetmol (China), 5FU (Cat#: F6627-1G) was
obtained from Sigma-Aldrich. Anti-p21 antibody (Cat#: 103551-1-AP)
and anti-p53 antibody (Cat#: 103551-1-AP) were from Proteintech (China).
Anti-γH2AX antibody (Cat#: ab11174) and anti-GAPDH antibody
(Cat#: ab181602) were from Abcam. Seven Color Protein Marker II (Cat#:
SW175-02) was obtained from Sevenbio (China). DMEM medium (Cat#: 01-052-1ACS)
and RPMI-1640 medium (Cat#: 01-100-1ACS) were from Biological Industries
(Israel). The fetal bovine serum (FBS) of Sijiqing (Cat#: 13011-8611)
was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (China).
120 small molecules was from the laboratory of Zhenyu Li. The phenylquinoline
derivatives were synthesized through the Suzuki coupling reaction
as in our previous description.33 (link) PFTα
(Cat#: T2707) was from Targetmol (China), 5FU (Cat#: F6627-1G) was
obtained from Sigma-Aldrich. Anti-p21 antibody (Cat#: 103551-1-AP)
and anti-p53 antibody (Cat#: 103551-1-AP) were from Proteintech (China).
Anti-γH2AX antibody (Cat#: ab11174) and anti-GAPDH antibody
(Cat#: ab181602) were from Abcam. Seven Color Protein Marker II (Cat#:
SW175-02) was obtained from Sevenbio (China). DMEM medium (Cat#: 01-052-1ACS)
and RPMI-1640 medium (Cat#: 01-100-1ACS) were from Biological Industries
(Israel). The fetal bovine serum (FBS) of Sijiqing (Cat#: 13011-8611)
was purchased from Zhejiang Tianhang Biotechnology Co., Ltd. (China).
4
Western Blot Analysis of Cell Cycle and Apoptosis Markers
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These specific operation steps were performed following the previous study with some modification (Sun et al., 2020 (link)). The specific binding sites were incubated with anti-Cycline D antibody (1:5000, Proteintech, USA), anti-Cyclin A antibody (1:500, Proteintech, USA), anti-Cyclin B antibody (1:1000, Proteintech, USA), anti-P53 antibody (1:500, Proteintech, USA), anti-Caspase 9 antibody (1:200, Proteintech, USA), anti-Caspase 8 antibody (1:100, Proteintech, USA), anti-Caspase 3 antibody (1:200, Proteintech, USA), anti-ILC3 antibody (1:1000, Proteintech, USA), anti-β-actin antibody (1:5000, Proteintech, USA), respectively. After incubation with anti-mouse/rabbit lgG HRP-linked secondary antibody (1:5000, Proteintech, USA), image and analysis were detected by ImageQuant LAS 4000 mini (GE Healthcare, China).
5
Quantitative Western Blot Analysis
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The total protein of the cells with different treatments was extracted using RIPA lysis buffer, and a BCA protein assay kit was used to measure the total protein concentrations. After that, the protein samples (20 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. After blocking with 5% skim milk at 37°C for 2 h, the membranes were incubated with anti-Axl antibody (1:1,000; Protein Tech Group, Inc.), anti-α-SMA antibody (1:1,000; Protein Tech Group, Inc.), anti-MMP3 antibody (1:1,000; Protein Tech Group, Inc.), anti-STAT4 antibody (1:1,000; Protein Tech Group, Inc.), anti-TIMP1 antibody (1:200; Santa Cruz, Inc.), anti-p53 antibody (1:2,000; Protein Tech Group, Inc.), anti- Caspase3 antibody (1:1,000; Protein Tech Group, Inc.), and anti-GAPDH antibody (1:10,000; Protein Tech Group, Inc.) at 4°C overnight. On the next day, the secondary antibody (goat anti-mouse IgG (H + L)-HRP [1:10,000; Jackson ImmunoResearch Laboratories, Inc.]) was added, and cells were incubated at 37°C for 2 h. Finally, the membrane was visualized using an ECL assay kit (Beyotime Institute of Biotechnology), and the protein expression levels of the related proteins were quantified using Image-Pro Plus software.
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