19. Determine the protein concentration with Bradford from the total aliquot (dilute 10, 5, 2.5, 1.25, 0.625 µl lysate into 10 µl HKM-Lysis buffer; and add 800 µl 1xBradford reagent; incubate 5 min at RT and measure at 595nm; make a fresh Bradford series with BSA (8,4,2,1,0.5 µg).
19. Determine the protein concentration with Bradford from the total aliquot (dilute 10, 5, 2.5, 1.25, 0.625 µl lysate into 10 µl HKM-Lysis buffer; and add 800 µl 1xBradford reagent; incubate 5 min at RT and measure at 595nm; make a fresh Bradford series with BSA (8,4,2,1,0.5 µg).
20. Prepare 3x6 tubes for the different reactions, with 1200 µl lysate each or less if some is lost somewhere
20. Prepare 3x6 tubes for the different reactions, with 1200 µl lysate each or less if some is lost somewhere
21. Calculate how much of your 15N bait protein you need to add (0.25:1, 0.5:1, 1:1, 2.5:1, 5:1, 10:1 15N:14N)
21. Calculate how much of your 15N bait protein you need to add, assuming CPN20 accounts for 0.17% of the total protein and CPN60A for 0.1% of the total protein (0.25:1, 0.5:1, 1:1, 2.5:1, 5:1, 10:1 15N:14N)
22. After the addition of the 15N protein take a 25 µl aliquot (freeze in -80°C)
22. After the addition of the 15N protein take a 25 µl aliquot (freeze in -80°C)
IP
IP
23. To each reaction add 100µl slurry, let the beads bind the epitopes for 1h at 4°C
23. To each reaction add 100µl slurry, let the beads bind the epitopes for 1h at 4°C
