The phosphoproteins from CAD and healthy control plasma were enriched using a phosphoprotein enrichment kit (Thermo Scientific, Pierce, USA) in triplicate using manufacturer's protocol as described earlier [22 (link)]. The columns were loaded with resin and equilibrated with exchange buffer. Plasma samples (CAD n = 5 and healthy n = 5) were pooled separately and used for phosphoprotein enrichment in order to minimize intraindividual heterogeneity. A total of 0.5-1.0 mg protein was taken from each pooled sample. The plasma samples were then exchanged with 0.5 M triethylammonium bicarbonate (TEAB, pH 8.5) buffer using 3 kDa cut-off filter (Millipore, USA). The fractions of phosphoproteins were eluted using elution buffer and quantitated by the Bradford assay.
Phosphoprotein enrichment kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Phosphoprotein Enrichment Kit is a laboratory tool designed to isolate and enrich phosphorylated proteins from complex biological samples. It utilizes affinity-based methods to selectively capture phosphorylated proteins, enabling their identification and analysis.
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Lab products found in correlation
2 protocols using phosphoprotein enrichment kit
1
Phosphoprotein Enrichment from Plasma
2
Phosphoprotein Enrichment from Brain Tissues
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From the other hemisphere, three brain parts CO, CS, and HI were dissected out and washed two times in 50 mM N-2hydroxyethylpiperazine -N′-2-ethanesulfonic acid (HEPES) buffer, pH 7.0. Tissue lysis was done according to the manufacturer's protocol (Phosphoprotein enrichment kit, Thermo Scientific, USA) with some modification. Briefly, for every 1 g of tissue 15 ml of Radio Immunoprecipitation Assay (RIPA) buffer having 1 X protease, Benzonase and phosphatases inhibitors were added. Preparation was homogenized and centrifuged at 10,000 × g for 5 min for removal of tissue debris. Supernatant was collected and protein estimation was done by Bradford method. 20 Final concentration was set to 0.5 mg/ml with Lysis/Binding/Wash buffer provided with the kit. Up to 4 mg of total protein preparation diluted with lysis/binding/wash buffer was used for phosphoprotein enrichment.
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