- Solubilize protein sample in 2X Sample buffer (Bio-Rad #1610737 or home made). Add DTT to 50 mM and heat at 65°C for 15 minutes.
- Load protein onto gel. Use a 6% or 7% polyacrylamide gel or better yet, a 4-15% polyacrylamide density. The amount of protein loaded will depend somewhat on the CFTR source material. Overexpressing cell lines require no more than 20 mg of tissue culture cell lysate or as little as 5 mg of a microsome prep.
- Transfer gel to nitrocellulose (I’ve had better luck with nitrocellulose than PVDF). I use the Bio-Rad TransBlot Turbo system. This is a semi dry transfer method and CFTR transfers in as little as 7 minutes from a 4-15% gel at 24V and 25A.
- Wash nitrocellulose in PBS for 5 minutes to remove transfer buffer
- Block nitrocellulose for a minimum of 1 hour to overnight using LiCor Intercept blocking buffer.
- Primary antibody concentration can vary depending on the amount and quality of material loaded on the gel. I start at 1:10000 dilution of ascites in block or LiCor antibody dilution buffer and incubate overnight at room temperature.
- Wash 3 x quickly with PBS and 1x 5 min PBST
- Secondary: 1:10000 Goat anti-Mouse 1 hour diluted in block or LiCor antibody dilution buffer
- Detect signal.
Some of this is intentionally vague since each lab has their own methods and detection systems. Consider this a guide and adjust for your own equipment.
PBS can be replaced with TBS
Some samples (like native tissues) may have a very low amount of CFTR and require immunoprecipitation to concentrate the sample enough to get a Western blot signal. Using antibodies of different isotypes for the IP and Western primary and an isotype specific secondary can prevent antibody signals from appearing in the Western blot.
2X Sample Buffer:
|0.5M Tris-HCl pH6.8
|1% Bromophenol blue