Protocol for Western & Immunoprecipitation of human CFTR using 3G11 rat monoclonal against mouse CFTR
The Scripps Research Institute
Department of Cell Biology, MB6
10550 N. Torrey Pines Road
La Jolla, CA 92037
Lysate is prepared as follows:
Lysis buffers: (A) Triton lysis buffer (TLB) (1% Triton, 25mM Tris pH 7.4, 150mM NaCl + protease tablets from Roche at 2 mg/ml).
1. Wash dishes with cold PBS.
2. Add lysis buffer to the dish, rock for about 30 min at 4°C. Typically we lyse in 2 ml for 150 mm dish, 833 ml for 100 mm dish, and 278 ml /60 mm dish.
3. Scrape lysate into a microfuge tube.
4. Spin 14,000 rpm (20,000 x g) for 20 min.
5. Transfer supernatant to a new tube.
6. Freeze aliquots in lysis buffer at –80°C, at 1.5-3 mg/ml.
1.Thaw aliquot on ice.
2. Add 5x Laemmli sample buffer with 125 mM DTT.
3. Heat at 37°C for 20 min. Note that CFTR precipitates and becomes insoluble if the sample is heated above 60°C and will not enter stacking gel.
4. Run 15-30 mg for wt and 45-90 mg for delta F on a 8% gel.
5. Transfer to nitrocellulose (we use 400mA for 90 min). Should migrate as a doublet at 140kDa (band B- weak band) and 160kDa (band C-strong band).
6. Block with TBS + 0.1% Tween 20 (TBS/T) with 4% milk.
7. Add 3G11 (rat monoclonal) in TBS + 0.1% Tween with 4% milk for 1 h at room temperature (RT) or 4°C overnight. See blot below for titration.
8. Wash 3 x 30 min with TBS/T.
9. Add Goat anti-Rat HRP (1:10, 000) in TBS/T for 1hr at RT. (SouthernBiotech, Alabama- Goat anti rat IgG H+L specific)
10. Wash 3x 30 min with TBS/T.
11. Develop with chemiluminescent substrates for HRP such as ECL Pico Western reagent (GE Healthcare or Pierce Supersignal Western Pico). See Figure 1.
HEKwt CFTR cells grown on 150 mm dishes are harvested ahead of time as follows:
1. Wash 150mm dishes with cold PBS.
2. Scrape in cold PBS.
3. Aliquot to 1.5 ml the equivalent of a 60 mm dish. (e,g., 7.2 samples/150 mm dish, 3 samples/100 mm dish, 1 sample/60mm dish).
4. Spin briefly in refrigerated microfuge tube to pellet cells (1 min, 20,000 x g).
5. Aspirate supernatant.
6. Freeze tubes at –80°C.
Thaw tubes containing cells on ice with the addition of 270 ml of lysis buffer/60 mm dish (protein concentration is about 1.5-3mg/ml).
(A) Triton lysis buffer (TLB) (1% Triton, 25 mM Tris pH 7.4, 150 mM NaCl + protease tablets from Roche at 2 mg/ml (important)).
(B) RIPA lysis buffer (RLB) (1% Triton, 150mM NaCl, 25mM Tris pH 7.4, 0.1% SDS, 0.5% deoxycholate + protease tablets (Roche) at 2 mg/ml).
Figure 1. Illustration of Western blotting (upper panel)
or immunoprecipitation (lower panel) of wild-type or DF508 from HEK293 cells. Protocol for IP and Western as outlined
above. In lower panel, immunoprecipitated CFTR was blotted using either
3G11 (left group) or M3A7 (right group). Representative lysate CFTR and bead control illustrated.
Figure 1. Illustration of Western blotting (upper panel) or immunoprecipitation (lower panel) of wild-type or DF508 from HEK293 cells. Protocol for IP and Western as outlined above. In lower panel, immunoprecipitated CFTR was blotted using either 3G11 (left group) or M3A7 (right group). Representative lysate CFTR and bead control illustrated.