The ovarian granulosa cells were lysed with TNE buffer (50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 0.5% NP-40). One microgram of antibody was added and incubated at 4°C for 12 h with gentle shaking. The protein A magnetic beads were added and incubated at 4°C for 1 h. Then, the beads were washed with TNE buffer five times and boiled at 100°C for 5 min. The granulosa cell samples of 3 PCOS patients and 3 controls were tested by western blotting with an anti-acetylated antibody (PTM-105, PTM Biolabs) to verify the changes of protein lysine acetylation, and the granulosa cell samples of 25 PCOS patients were examined to analyze the correlation of the lysine acetylation of ACAT1 protein to the clinical outcomes. Antibodies included PGK1 (17811-1-AP, Proteintech), PGAM1 (ab129191, Abcam), GAPDH (ab181602, Abcam), and ACAT1 (#44276, Cell Signaling Technology).
Ptm 105
Manufactured by PTM Biolabs
The PTM-105 is a laboratory instrument designed for the detection and analysis of post-translational modifications (PTMs) in proteins. It utilizes advanced chromatographic techniques and mass spectrometry to identify and quantify various types of PTMs, such as phosphorylation, acetylation, and glycosylation, in complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ptm 401, by PTM Biolabs (2 mentions)
Ab129191, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Anti stat1, by Cell Signaling Technology (1 mentions)
Anti β actin sc 5286, by Santa Cruz Biotechnology (1 mentions)
Ab1791, by Abcam (1 mentions)
Ptm 501, by PTM Biolabs (1 mentions)
Ptm 419, by PTM Biolabs (1 mentions)
Ptm 301, by PTM Biolabs (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Anti sirt7, by Santa Cruz Biotechnology (1 mentions)
Anti ku80, by Santa Cruz Biotechnology (1 mentions)
Anti parp1 2, by Santa Cruz Biotechnology (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
4 protocols using ptm 105
1
Lysine Acetylation Analysis in PCOS
2
Immunoprecipitation and Western Blotting of STAT1 Acetylation
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Mouse lung tissue samples were lysed in RIPA buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol) with protease/phosphatase inhibitor cocktail (5872S, Cell Signaling Technology, 1:1000), 0.3 mM DTT, 5 mM nicotinamide, and 1 mM sodium butyrate. Total 500 μg of protein were used for immunoprecipitation with indicated dilution of each antibody (anti-STAT1, 14994, Cell Signaling Technology, 1:100).
Western blotting was performed according to standard protocols. The antibodies and dilutions were anti-β-actin (sc-5286, Santa Cruz, 1:10,000), anti-STAT1 (14994, Cell Signaling Technology, 1:1000), anti-acetyl-lysine (PTM-105, PTM Bio, 1:1000), and anti-succinyl-lysine (PTM-401, PTM Bio, 1:1000).
Western blotting was performed according to standard protocols. The antibodies and dilutions were anti-β-actin (sc-5286, Santa Cruz, 1:10,000), anti-STAT1 (14994, Cell Signaling Technology, 1:1000), anti-acetyl-lysine (PTM-105, PTM Bio, 1:1000), and anti-succinyl-lysine (PTM-401, PTM Bio, 1:1000).
3
ChIP-Seq Profiling of Histone Modifications
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Epi-ID was performed as described23 (link), 25 (link), except that for ChIP the following antibodies were used: anti-pan-K-acetylation (PTM-105; PTM-Biolabs), anti-pan-K-crotonylation (PTM-501;PTM Biolabs), anti-pan-K-butyrylation (PTM-301; PTM Biolabs), anti-pan-K-succinylation (PTM-419; PTM Biolabs) and anti-H3 (ab1791; Abcam). Deep-sequencing was performed on a single-end flowcell Illumina Hi-Seq2500.
4
Comprehensive Protein Analysis by Western Blot
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Western blotting was performed according to standard procedures. Antibodies used were anti-SIRT7 (Santa Cruz Biotechnology, sc-365344, 1:500), anti-HA (MBL, M180-3, 1:2,000), anti-Flag (Sigma, F3165, 1:10,000), anti-β-actin (Sigma, A1978, 1:10,000), anti-tubulin (Sigma, clone B-5-1-2, T6074, 1:50,000), anti-SIRT6 (Abgent, AP-6245a, 1:500), anti-PARP1/2 (Santa Cruz Biotechnology, sc-7150, 1:5,000), anti-Ku80 (Santa Cruz Biotechnology, sc-5280, 1:2,000), anti-BRCA1 (Proteintech, 22362-1-AP, 1:1,000), anti-γH2AX (Millipore, 05-636, 1:2,000), anti-H2AX (Abcam, ab11175, 1:2,000) anti-pan-acetylation (PTM BioLabs, PTM-105, 1:1,000), anti-pan-succinylation (PTM BioLabs, PTM-401, 1:1,000), anti-H3K122succ (1:4,000), anti-H2BK120succ (1:8,000), anti-H3K122ac (Abcam, ab33309, 1:2,000), anti-H3K18ac (PTM BioLabs, PTM-114, 1:1,000), anti-H3 (Abcam, ab1791, 1:100,000) and anti-rabbit (Jackson ImmunoResearch, 115-035-003, 1:8,000) or anti-mouse (Jackson ImmunoResearch, 111-035-003, 1:8,000) secondary antibodies conjugated to horseradish peroxidase. The bands were quantified by densitometry with ImageJ software. Uncropped scans of the most important blots are shown in Supplementary Fig. 11 .
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