Western Blotting Protocol (Primary Ab Incubation In Milk)

For Western blots, incubate membrane with diluted antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween-20 at 4°C with gentle shaking, overnight.

Products available from Cell Signaling Technology are linked by their respective catalog numbers.

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 1X Phosphate Buffered Saline (PBS).
2. 1X SDS Sample Buffer: (#7722, #7723) 62.5 mM Tris-HCl (pH 6.8 at 25°C), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue or phenol red.
3. Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol (pH 8.5).
4. 10X Tris Buffered Saline (TBS): (#9997) To prepare 1 liter of 10X TBS: 24.2 g Tris base, 80 g NaCl; adjust pH to 7.6 with HCl (use at 1X).
5. Nonfat Dry Milk: (#9999) (weight to volume [w/v]).
6. Blocking Buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk; for 150 ml, add 15 ml 10X TBS to 135 ml water, mix. Add 7.5 g nonfat dry milk and mix well. While stirring, add 0.15 ml Tween-20 (100%).
7. Wash Buffer: 1X TBS, 0.1% Tween-20 (TBS/T).
8. Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween-20 with 5% nonfat dry milk; for 20 ml, add 2 ml 10X TBS to 18 ml water, mix. Add 1.0 g nonfat dry milk and mix well. While stirring, add 20 μl Tween-20 (100%).
9. Phototope^®-HRP Western Blot Detection System: (#7071 anti-rabbit) or (#7072 anti-mouse) Includes biotinylated protein ladder, secondary (#7074 anti-rabbit) or (#7076 anti-mouse) antibody conjugated to horseradish peroxidase (HRP), anti-biotin antibody conjugated to HRP, LumiGLO chemiluminescent reagent and peroxide.
10. Prestained Protein Marker, Broad Range (Premixed Format): (#7720).
11. Biotinylated Protein Ladder Detection Pack: (#7727).
12. Blotting Membrane: This protocol has been optimized for nitrocellulose membranes, which CST recommends. PVDF membranes may also be used.

B. Protein Blotting

A general protocol for sample preparation is described below.

1. Treat cells by adding fresh media containing regulator for desired time.
2. Aspirate media from cultures; wash cells with 1X PBS; aspirate.
3. Lyse cells by adding 1X SDS sample buffer (100 μl per well of 6-well plate or 500 μl per plate of 10 cm diameter plate). Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice.
4. Sonicate for 10–15 seconds for complete cell lysis and to shear DNA (to reduce sample viscosity).
5. Heat a 20 μl sample to 95–100°C for 5 minutes; cool on ice.
6. Microcentrifuge for 5 minutes.
7. Load 20 μl onto SDS-PAGE gel (10 cm x 10 cm).

NOTE: CST recommends loading prestained molecular weight marker (#7720, 10 μl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 μl/lane) to determine molecular weights.

1. Electrotransfer to nitrocellulose or PVDF membrane.

C. Membrane Blocking and Antibody Incubations

NOTE: Volumes are for 10 cm x 10 cm (100 cm²) of membrane; for different sized membranes, adjust volumes accordingly.

1. (Optional) After transfer, wash nitrocellulose membrane with 25 ml TBS for 5 minutes at room temperature.
2. Incubate membrane in 25 ml of blocking buffer for 1 hour at room temperature.
3. Wash three times for 5 minutes each with 15 ml of TBS/T.
4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C.
5. Wash three times for 5 minutes each with 15 ml of TBS/T.
6. Incubate membrane with appropriate HRP-conjugated secondary antibody (1:2000) and HRP-conjugated anti-biotin antibody (1:1000) to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hour at room temperature.
7. Wash three times for 5 minutes each with 15 ml of TBS/T.

D. Detection of Proteins

1. Incubate membrane with 10 ml LumiGLO (0.5 ml 20X LumiGLO, 0.5 ml 20X Peroxide and 9.0 ml Milli-Q water) with gentle agitation for 1 minute at room temperature.

NOTE: LumiGLO substrate can be further diluted if signal response is too fast.

1. Drain membrane of excess developing solution (do not let dry), wrap in plastic wrap and expose to x-ray film. An initial 10-second exposure should indicate the proper exposure time.

NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following LumiGLO incubation and declines over the following 2 hours.