From 3913a89a5c5b0fcb80cfdc8eb3426153c06de1e5 Mon Sep 17 00:00:00 2001 From: Fabian Ries <riesf@rhrk.uni-kl.de> Date: Fri, 16 Jun 2023 11:26:01 +0000 Subject: [PATCH] information added for calculation of competitor amount --- studies/competition co-IP/protocols/coIP.txt | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/studies/competition co-IP/protocols/coIP.txt b/studies/competition co-IP/protocols/coIP.txt index a94dd4858..20c992bc0 100644 --- a/studies/competition co-IP/protocols/coIP.txt +++ b/studies/competition co-IP/protocols/coIP.txt @@ -27,7 +27,7 @@ Cell harvest: 18. Ensure that your lysate is homogenous 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 -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) IP 23. To each reaction add 100µl slurry, let the beads bind the epitopes for 1h at 4°C -- GitLab