Protocol for Western & Immunoprecipitation of human CFTR using 3G11 rat monoclonal against mouse CFTR

 

Balch Lab

The Scripps Research Institute

Department of Cell Biology, MB6

10550 N. Torrey Pines Road

La Jolla, CA 92037

 

 

Preparation of Lysates for Western Blot of HEK293 cells expressing wild-type hCFTR or DF508CFTR

 

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 4C. 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 80C, at 1.5-3 mg/ml.

 

Western Blot Protocol

1.Thaw aliquot on ice.

2. Add 5x Laemmli sample buffer with 125 mM DTT.

3. Heat at 37C for 20 min. Note that CFTR precipitates and becomes insoluble if the sample is heated above 60C 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 4C 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.

 

Preparation of Cells for Immunoprecipitation

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 80C.

 

Immunoprecipitation Protocol

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).

Lysis buffers:

(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).

 

  1. After addition of lysis buffer, leave on ice for about 10-15 min.
  2. Spin 14,000 rpm (20,000 x g) for 20 min.
  3. To supernatant add 30 ml of 50% washed protein G beads to pre-clear the lysate.
  4. Incubate ~60 min at 4C with rocking.
  5. Spin out beads (20 s top speed in microfuge).
  6. Spin at 100,000 x g in the ultracentrifuge at 4C for 20 min to remove insoluble aggregates.
  7. Transfer supernatant to a 1.5 ml tube containing 25-35ul of a 50% suspension of 3G11 beads covalently coupled at 4mg/ml to protein G beads. (Alternatively- add Ab separately for 45 min then add protein G beads overnight - we have not tested this condition and would need to determine how much antibody to add. We usually start testing at 10 mg/ml of Ab.)
  8. Incubate overnight with rocking at 4C.
  9. Wash 2X with TLB and 1X with TLB minus detergent.
  10. Add 14 ml of 2X gel sample buffer (Laemmli) containing 50 mM DTT.
  11. Heat at 37C for 20 min. Note that CFTR precipitates and becomes insoluble if the sample is heated above 60C and will not enter gel.
  12. Run 8% gel and transfer to nitrocellulose. Should migrate as a doublet at 140kDa (band B- weak band) and 160kDa (band C-strong band)
  13. Western was performed as described above in Western protocol (steps 6-11). As illustrated below, used either 3G11 at 2 mg/ml or M3A7 (Ascites) at 1:2500 was used in TBS/T/milk. Goat anti rat or goat anti mouse are 1:10,000 in TBS/T. See blot for more information. See Figure 1.

 

 

 

 

 

 

 

 

 

 

 

 

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.

 
 

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