Total protein was extracted from EC cells and tissues through phenylmethylsulfonyl fluoride and protease inhibitors according to the instructions. After the samples had been lysed at 4 °C for 15 min and centrifuged at 15,000 r/min for 15 min, a bicinchoninic acid kit (23227, Thermo Fisher Scientific Inc.) was used to provide an estimation of the protein concentration within the supernatant. After the protein had been separated by polyacrylamide gel electrophoresis methods, the protein on the gel was electroblotted to a polyvinylidene fluoride membrane and subsequently blocked by 5% bovine serum albumin (BSA) at room temperature for 1 h. The membrane was subsequently probed overnight at 4 °C with the diluted primary rabbit antibodies (Abcam Inc.) against p53 (0.5 μg/mL, ab183544), FOXP3 (10 μg/mL, ab215206) and GAPDH (0.5 μg/mL, ab181602). The membrane was then re-probed with horseradish peroxidase labeled goat anti-rabbit IgG secondary antibody (0.1 μg/mL, ab205718, Abcam Inc.) at room temperature for 1.5 h. Developing solution (NCI4106, Pierce, Rockford, IL, USA) was subsequently added to the membrane for color development purposes. Image J 1.48u software (Bio-Rad, Hercules, CA, USA) was employed for protein quantitative analysis, which was expressed as the ratio of the gray value of each protein to that of the internal reference.
Ab183544
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab183544 is a laboratory product manufactured by Abcam. It is a tool used for research purposes, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information about the intended use or specific details of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab7780, by Abcam (2 mentions)
Ab215206, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Imagej 1.48u software, by Bio-Rad (1 mentions)
Bicinchoninic acid kit, by Thermo Fisher Scientific (1 mentions)
Ab8895, by Abcam (1 mentions)
Ab12188, by Abcam (1 mentions)
Ab70378, by Abcam (1 mentions)
Ab8898, by Abcam (1 mentions)
Ab10812, by Abcam (1 mentions)
Ab37415, by Abcam (1 mentions)
Ab2893, by Abcam (1 mentions)
Alexa fluor 555 goat anti rabbit ab150078, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse ab150117, by Abcam (1 mentions)
Rabbit anti mouse ab6728, by Abcam (1 mentions)
Hoechst 43222 h1399, by Thermo Fisher Scientific (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab1791, by Abcam (1 mentions)
Penicillin, by Merck Group (1 mentions)
5 protocols using ab183544
1
Protein Extraction and Quantification Protocol
2
Chromatin Modifications in MEF Cells
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All cell lines were cultured in DMEM medium with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (Millipore-Sigma) in a humidified incubator with 5% CO2 at 37 °C.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos46 (link). In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized2 (link). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.
Initial experiments were performed on primary MEFs derived from 13.5 days NM1 WT and KO embryos46 (link). In other experiments were used immortalized MEFs (ATCC® CRL-2752) or cell lines derived from these immortalized cells.
The antibodies against H3K9me3 (ab8898), H3K27ac (ab4729), H3K4me1 (ab8895), H3K4me3 (ab8580), H3K9ac (ab10812), H3 (ab1791), γH2AX (ab2893), p53-K370 (ab183544), PCAF (ab12188), Set1 (ab70378), GAPDH (ab8245), and nonspecific rabbit IgG isotype control (ab37415) were purchased from Abcam (Cambridge, MA, USA). The antibody against NM1 has been previously characterized2 (link). Alexa Fluor 555 Goat Anti-Rabbit (ab150078) and Alexa Fluor 488 Goat Anti-Mouse (ab150117) were used as secondary antibodies for immunofluorescence, and horseradish peroxidase (HRP)-fused Goat Anti-Rabbit (ab6721) and Rabbit Anti-Mouse (ab6728) secondary antibodies for western blottings were purchased from Abcam. Hoechst 43222 (H1399) and ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole (DAPI; P36931) were purchased from Invitrogen, Waltham, MA, USA. All antibodies have been used according to the manufacturers’ protocols.
3
Protein Expression Analysis of Gastrocnemius Muscle
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The frozen gastrocnemius muscle samples were homogenized in a radioimmunoprecipitation assay buffer containing 1 mM phenylmethylsulfonyl fluoride and the Protease Inhibitor Cocktail (Roche Applied Science). The lysates were centrifuged for 20 min at 12000 × g (4 °C), and the protein level in the supernatant was quantified with a bicinchoninic acid assay kit (Beyotime). The proteins were separated by SDS–PAGE (Beyotime) and transferred to a polyvinylidene difluoride membrane (Beyotime) that was blocked with 5% non-fat dry milk in tris-buffered saline at room temperature, followed by incubation with primary antibodies: mouse anti-progerin (1:200; Santa Cruz Biotechnology, Sc-81611, USA), rabbit anti-α-SMA antibodies (1:1000; Affinity Biosciences, AF1032, USA), rabbit anti-P53 (1:1000; Abcam, Ab32532, UK) and anti-acetyl-P53 (1:1000; Abcam, Ab183544, UK), and rabbit anti-iNOS antibodies (1:1000; Affinity Biosciences, AF0199, USA). After being washed three times, the membrane was incubated with the appropriate secondary antibody (Abcam) at room temperature for 1 h. The enhanced chemiluminescence detection reagent and an X-ray film were used to visualize the proteins.
4
Immunoprecipitation and Western Blot Analysis
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After a 48 h period of transfection, the cells were placed on ice. IP lysis solution was added to the cells in each well after the medium had been removed. The cells were scraped using a cell scraper, and spread evenly using a pipette followed by transfer to an eppendorf (EP) tube. The cells were subsequently lysed on ice for 30 min, followed by centrifugation at 800 rpm and 4 °C for 20 min. The supernatant was then transferred into a new EP tube, with the protein concentration determined using a bicinchoninic acid (BCA) method. The 1 mg protein in each sample was brought to 500 μL with IP lysis solution. Next, 500 μL of each sample was added with primary antibodies and incubated overnight at 4 °C on a mute mixer, respectively. Each tube was supplemented with 20 μL protein A + G beads in the morning of the next day, followed by culture for 2 h. After the impurities had been eluted with IP lysis, the samples were centrifuged at 2500 rpm and 4 °C for 5 min, and washed 5 times. The supernatant was discarded following centrifugation. Next, 20 μL of 2 × loading buffer was assed to each tube and subsequently denatured in metal bath at 100 °C for 5 min. The samples bound to IP were then subjected to western blot analysis using antibodies to p53 (0.5 μg/mL, ab183544, Abcam Inc.) and anti-Ubiquitin (1 μg/mL, ab7780, Abcam Inc.).
5
Immunoprecipitation of Ubiquitinated p53
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MUM-2B/CDDP cells were lysed on ice with IP lysate containing protease inhibitor MG-132. Next, 1 mg protein was harvested from each sample, the volume of which was made equal to that of IP lysate. p53 monoclonal antibody was added to the cells for IP, followed by incubation at 4 °C overnight. In the morning of the next day, 20 μL protein A + G beads were added to the cells for incubation for 2 h. After centrifugation, the supernatant was removed carefully, followed by addition of 2 × loading buffer (20 μL/well). The gained samples were adopted for subsequent sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot assay. The antibodies from Abcam utilized in this test were anti-IgG (1: 100, ab172730), anti-Ub (1: 100, ab7780), and anti-p53 (1: 100, ab183544).
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