This protocol utilises an Anti HA Ab, but one can use any other Ab depending to what you want to IP.
Remember to standerize the IP (ammount of Ab used)
CO-IP of membrane protein and HSP complex (Guy June 2006)
Purpose: To CO-IP HSP's with membrane protein in different BHK CFTR cell lines
Amersham G-beads (fast flow) is used
PBS Ice cold supplemented with 1mMMg and 0.1mMCa (PBSMC)
Mild Lysis Buffer - 150mM NaCl, 20mM Tris HCl, 0.1% NP-40, pH 7.4) Supplemented with Protease inhibitors 10ug/ml Leupeptin, pepstutin, 1mM PMSF and 10mM Iodoacetamide.
Anti HA mA Hybridoma 1:20 (200ul per 5ml Lysate)
Anti HA COVANCE MMS 101Raw ascites (10ul per 5ml Lysate)
Protein G-beads sepharose 4 fast flow (GE) lot309778 cat# 17-0618-01
Pre clean beads with lysate buffer 3 times)
15CM dish for each cell line - mutated CFTR 3 dishes, WT 2 dishes, BHK 3 dishes.
1. Transfer BHK cells from 1 10cm to 15cm 2 days before beginning rescue or IP.
2. Wash with PBSMC the 15 cm dishes
3. Scarpe cells in 15ml PBS, pellet at 2500rpm for 5min at Beckman countertop.
4. Solobilise with Mild lysis buffer for 8 minutes on Ice
5. Spin down post nuclear ppellet at 4800rpm for 10 minutes, beckman
6. Take 200ul from lysate to clean tube (protein concentration)
7. PreClean Lysate with 50ul 50% G-bead slurry for 30 min rotating at 4°C
8. Add 200ul Hybridma anti HA, 10ul Covance anti HA, rotate 1hour at 4°C
9. Add pre 140ul 50% slurry of pre-cleaned G-Beads rotate 1hour at 4°C
10. Wash 3 times with 15 ml lysis buffer, spin down beads at 1000rpm for 2min , Beckman
11. Elute with X2 LSB (supplemented with 10% b-ME) for 20 min at 37°C
12. Separate on 11% Gel for HSP's and 7% Gel for HA
Calculations: Calculate the ammount of CO-IP as a precentage of the total of HA in 100ug total prtein
One can use any Ab for the IP