Method of detecting protein palmitoylation modification locus
A detection method and palmitoylation technology, applied in the field of protein post-translational modification detection, can solve the problems of difficult to purify protein samples, limited analysis samples, complicated operations, etc. stable effect
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[0046] 1Purification of target protein
[0047] 1.1 Sample preparation
[0048] The APT1 protein with Flag tag was overexpressed in HEK-293T cells, and the Flag tag encoded a hydrophilic polypeptide DYKDDDDK of 8 amino acids. The protein was extracted with Beyond P0013IP cell lysate to obtain APT1 protein solution, the measured concentration was 3mg / ml, and 30mg of the protein solution was taken.
[0049] 1.2 Purified protein
[0050] Mix the anti-DYKDDDDK tag affinity medium (Sino Biological, Catalog: 101274-MM13-RN) upside down, take 150μl medium and add 15ml containing 3ml equilibration buffer (10mMPBS, pH 7.4) In a centrifuge tube, centrifuge at 1000g for 3 minutes (washing medium); repeat washing twice with 3ml of equilibration buffer (avoid taking out the medium); add about 10ml of target protein solution to the washed medium, and place it on a rotator at 4°C overnight; The protein solution was centrifuged at 1000g for 3min, and the supernatant was removed; the precip...
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